Use of slurp1 as an imunomodulatory molecule in the ocular surface

ABSTRACT

Methods for treating inflammation are disclosed, such as for treating ocular inflammation. In some embodiments, the ocular inflammation is inflammation of an ocular surface, such as keratitis. The methods include administering to a subject with inflammation a therapeutically effective amount of SLURP1, or a nucleic acid encoding SLURP1, thereby treating the inflammation.

PRIORITY CLAIM

This claims the benefit of U.S. Provisional Application No. 61/722,712,filed Nov. 5, 2012, which is incorporated herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. NIHEY016875, NIH EY 10359, and NIH P30EY08098 awarded by the National EyeInstitute of the National Institutes of Health. The government hascertain rights in the invention.

FIELD

This disclosure relates to the field of inflammatory disorders,specifically to the use of Slurp1 and agonists thereof for the treatmentof ocular inflammation.

BACKGROUND

Injury, infection and allergy of the eye stimulate inflammatoryreactions. Humoral and cellular immunity are involved in many externaleye diseases. Inflammatory corneal disease can be infectious ornon-infections, and can be vision threatening. The infectious causes ofocular inflammation are numerous, and include bacteria, viruses, fungiand parasites. The initial events of exogenous ocular infections involvemicrobial adherence, invasion and multiplication. Recurrent viralinfections can also cause ocular inflammation.

The initial stages of ocular inflammation, such as inflammation of asurface of the eye, are often non-specific. These early manifestationsinclude pain, warmth, redness and swelling. Common symptoms ofinflammatory disease of the outer eye are itching, discomfort, dryness,redness, tearing, discharge, and blurred vision.

The most common cause of redness in the eye is conjunctivalinflammation. Common causes of conjunctivitis include papillaryconjunctivitis, follicular conjunctivitis, conjunctival granuloma andconjunctival ulceration. Keratitis is inflammation of the cornea. Commoncauses of keratitis are corneal injury, dry eye syndrome, viralinfections such as adenovirus, herpes simplex, and varicella-zosterinfections, bacterial infections, fungal infections, and autoimmunedisorders. There is a need for methods for treating ocular inflammation,including keratitis and conjunctivitis.

SUMMARY

Methods for treating ocular inflammation are disclosed herein. In someembodiments, the methods include administering to a subject with ocularinflammation a therapeutically effective amount of Slurp1, or a nucleicacid encoding Slurp1, thereby treating the inflammation.

In some embodiments, methods are disclosed for treating the inflammationof an external surface of the eye of a subject, such as the corneal orconjunctiva, or intraocular inflammation of the anterior or posteriorchamber of the eye. In particular embodiments, methods are disclosed fortreating corneal inflammation such as keratitis, intraocularinflammation such as uveitis or conjunctival inflammation such asconjunctivitis in a subject.

In further embodiments, the methods include selecting a subject withocular inflammation, and administering to the subject a therapeuticallyeffective amount of Slurp1 polypeptide, or a polynucleotide encoding theSlurp1 polypeptide, thereby treating the subject. In some specificnon-limiting examples, the subject is human. In additional non-limitingexamples, the ocular inflammation is keratitis, such as keratitis causedby non-traumatic etiologies such as an infection, for example viral orbacterial infection, or inflammation caused by non-infectious means,such as trauma.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. corneal expression of Slurp1. A. QPCR demonstratingpost-eyelid opening increase in Slurp1 expression Slurp1 expressionincreases more than 15-fold between post-natal day 11 (PN11) and PN21 B.Immunofluorescent staining of PN11, PN21 and PN56 mouse corneas showingelevated expression of Slurp1 (red) in corneal epithelium in post-eyelidopening stages. C. Immunofluorescent staining demonstrating expressionof SLURP1 (green) in human corneas. Post-mortem corneal sections from ahealthy 52 year-old male organ donor were used. Nuclei are stained withDAPI (blue) and corresponding ‘no antibody controls’ are shown in B andC. Signals emanating from the Descemet's membrane (in panel C-iv) appearto be due to autofluorescence, as they were detected in no primaryantibody controls (panel C-iii) as well. Scale bars: 25 μm in B and 50mm in C.

FIGS. 2A-2D. Downregulation of Slurp1 expression in the Klf4CN cornea.A, Changes in Klf4 expression during mouse corneal development. Absolutenumbers of Klf4 transcripts per ng total RNA were calculated using thestandard curve method of QPCR with total RNA from mouse corneas atdifferent stages of development. B, Slurp1 transcript levels in the WTand Klf4CN corneas measured by microarray (Swamynathan et al., InvestOphthalmol Vis Sci 2008; 49:3360-3370) and QPCR. C, Immunoblot withrabbit anti-mouse Slurp1 antibody detects a strongly reacting band atabout 21 kDa in the WT, but not in Klf4CN corneal extracts (left panel).The blot was stripped of the primary antibody and re-probed withanti-actin antibody, to ensure equal loading of protein (right panel).D, Immunofluorescent staining with anti-Slurp1 antibody. Left panel, WTwith no primary antibody; middle panel, WT with anti-Slurp1 antibody;Right panel, Klf4CN with anti-Slurp1 antibody.

FIG. 3A-3F. Klf4 binds and stimulates Slurp1 promoter activity. A,Schematic representation of the reporter vectors used. B, C, Relativepromoter activities of different sized Slurp1 promoter fragments withincreasing amounts (0, 100 or 500 ng) of co-transfected pCI-Klf4, in HCE(B) and NCTC(C) cells. D, Effect of siRNA-mediated knockdown of KLF4 on−500/+27 bp Slurp1 promoter activity in HCLE cells. Slurp1 promoteractivity was reduced upon knockdown of KLF4 expression by two differentsiRNAs, relative to that obtained with co-transfection of control siRNAexpressing plasmid. E, Chromatin immunoprecipitation was performed usingHCE cells and anti-KLF4 antibody. PCR amplified SLURP1 proximal promoterfragments from the input chromatin (lanes 1-2) or immunoprecipitatedchromatin (lanes 3-4) are shown. Lane 3, mock immunoprecipitated with noantibody; lane 4, immunoprecipitated with anti-KLF4 antibody. F,Nucleotide sequence of SLURP1 proximal promoter (SEQ ID NO: 7).Potential KLF4-interacting elements are shown underlined. Transcriptionand translation start sites are indicated.

FIGS. 4A-4D. Localization of CD45⁺ cells in the WT and Klf4CN corneas.Flat mounts of WT (panels A and C) and Klf4CN (panels B and D) corneaswere stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD45antibody and examined by confocal microscopy. Representative stackedimages of the central region of corneas are shown at 20× (panels A andB; numerical aperture (NA) 0.85) and 60× (panels C and D; NA 1.42)magnification. Note the relatively even distribution and lower densityof CD45⁺ cells in the WT corneas compared with their higher density andclustering in Klf4CN corneas.

FIGS. 5A-5C. Expression of Klf4 and Slurp1 in herpes simplex virusserotype-1 (HSV-1) infected (A and B), and (C) bacteriallipopolysaccharide (LPS)-injected corneas. Control (mock infected orPBS-injected), HSV-1 infected (A), or LPS-injected (C) WT mouse corneaswere harvested at the indicated time after treatment. Klf4 and Slurp1transcripts were quantified by QPCR. Bars indicate relative expressionlevels (mean±SEM) of Klf4 and Slurp1 in HSV-1 infected (A), orLPS-injected (C) corneas. B. Immunofluorescent staining with anti-Slurp1antibody in mock- or HSV-1-infected mouse corneas at 1 and 2 dayspost-scratching (DPS, Control) or post-infection (DPI). Nuclei arestained with DAPI (blue) and corresponding ‘no antibody controls’ areshown. Note that Slurp1 is abundantly expressed in control (panels iiiand iv), and sharply decreased in HSV-1 infected corneal epithelia(panels v and vi) at both 1 and 2 DPI. Scale bars: 25 mm.

FIGS. 6A-6D. Leukocyte populations in mock- and HSV-1-infected corneas.Mock- or HSV-1-infected corneas from WT and Klf4CN littermates wereexcised 2 DPI, the cells dispersed with collagenase, stained withfluorochrome-conjugated antibodies specific for CD45, CD11b, and Gr-1,and analyzed by flow cytometry. (A) Representative dot plots illustrategating on CD45⁺ cells. Percentage of CD45⁺ cells among stromal cells isshown within each dot plot. (B) The scatter plot shows the frequency ofCD45⁺ cells in mock infected and HSV-1 infected corneas. (C)Representative dot plots illustrate gating on CD11b⁺ Gr-1⁺ cells withina population gated on CD45⁺ cells. Percentage of Gr-1⁺ CD11b⁺ cellsamong stromal CD45⁺ cells is shown within the upper right quadrant ofeach dot plot. (D) The scatter plot shows the frequency of CD11b⁺ Gr-1⁺cells among CD45⁺ cells in mock infected and HSV-1 infected corneas.

FIGS. 7A-7D. Interferon-gamma (IFN-γ) is not required for Slurp1down-regulation and neutrophilic infiltration into infected corneas. (A)Slurp1 and Klf4 transcripts in HCLE cells exposed to differentcytokines. QPCR was performed with total RNA from HCLE cells exposed todifferent cytokines indicated for two days, with 18s rRNA as endogenouscontrol. Mean data from 3 independent experiments is presented. Errorbars represent standard error of mean (SEM). The p values werecalculated using Student's t-test. (B) Slurp1 and Klf4 transcripts inmock- or HSV-1 infected BALB/c WT or GKO corneas at 2 DPI. Bars indicatethe mean±SEM fold change in Slurp1 expression in mock- or HSV-1 infectedcorneas over that in control corneas. The p values were calculated usingStudent's t-test. (C & D) The cells in mock- or HSV-1 infected WT or GKOcorneas were dispersed with collagenase, stained withfluorochrome-conjugated antibodies specific for CD45, CD11b, and Gr-1,and analyzed by flow cytometry. (C) The scatter plot shows the frequencyof CD45⁺ cells in mock infected and HSV-1 infected corneas. (D) Thescatter plot shows the frequency of CD11b⁺ and Gr-1⁺ double positivecells among CD45⁺ cells in mock infected and HSV-1 infected corneas.Cumulative data from five different abraded mock- or HSV-infected WT andGKO animals each is shown.

FIGS. 8A-8D. Evidence for immunomodulatory role for Slurp1. WT BALB/cmouse corneas (n=4) abraded and infected with either 2×10⁶ PFUAdv5-Tet-Off vector alone (a, b, e, f) or 10⁶ PFU each of Adv5-Slurp1and Adv5-Tet-Off vectors (c, d, g, h) were imaged 4 and 10 DPI undernormal (a, e, c, g) and slit-lamp (b, f, d, h) illumination (A). Signsof mild inflammation were observed in corneas infected with Adv5-Tet-Offvector alone, while those infected with Adv5-Slurp1 and Adv5-Tet-Offvectors remained normal (A). Corneas harvested at 4 DPI were separatedinto epithelium and stroma+endothelium. Total RNA isolated fromepithelial cells was used to quantify relative expression of Slurp1 byQPCR (B). Stromal cells were isolated and stained withfluorochrome-conjugated antibodies specific for CD45, CD11b and Gr-1,and analyzed by flow cytometry (C and D). Number of CD45+ (C) and CD11b⁺Gr-1⁺ cells (D) was significantly reduced in Adv5-Slurp1 infectedcorneas compared with those infected with Adv5-Tet-Off vector (control)alone.

FIGS. 9A-9B. Proposed model for the function of Slurp1 in the ocularsurface. Slurp1 is a part of the machinery that suppresses inflammationat the ocular surface. A. Under normal conditions or conditions of mildtrauma, Klf4 supports constitutive high level production of Slurp1 bythe corneal epithelium. Slurp1 can (i) block urokinase-type plasminogenactivator receptor (uPAR) function by competing for its ligandsurokinase-type plasminogen activator (uPA), vitronectin (Vn) andintegrins (Intg), and/or (ii) interact with membrane-bound nicotinicacetylcholine receptor (nAchR) on the surface of resident corneal cellssuch as macrophages and dendritic cells, potentiating the ability ofacetylcholine (bound to nAchR) to block the release of intracellularTNF-α, maintaining the cornea in a non-inflamed state. B. Under severetrauma or microbial infection favoring inflammation, Slurp1 productionis rapidly reduced by pro-inflammatory cytokines, facilitating (iii)uPAR-mediated extracellular matrix (ECM) degradation and/or (iv) therelease of intracellular cytokines such as TNF-α, culminating inneutrophilic infiltration.

FIG. 10. Effect of Slurp1 on mouse corneal fibroblast MK/T-1 cellproliferation. MK/T-1 cells were seeded in a tissue culture-treated 96well plate. One set was removed for staining 4 hours after plating (0h). For the rest, Slurp1 was added at 0, 0.1 or 1 mg/ml in 100 μl mediumwith 5% FBS. Fresh medium with/without Slurp1 was added at 24 h intervalas required. Cells were fixed and stained with crystal violet atappropriate times and absorbance measured at 590 nm. The presence ofSlurp1 decreased the rate at which MK/T-1 cells proliferated.

FIG. 11. Effect of Slurp1 on adhesion of MK/T-1 cells to collagen-I-,-IV- and fibronectin-coated surfaces. MK/T-1 cells were harvested using0.4 mM EDTA (Trypsin was not used to avoid digestion of membrane-boundproteins). The cells were incubated for 1 h at 37° C. with increasingamounts of Slurp1 on collagen-I, collagen-IV, or fibronectin coatedplates blocked with BSA, washed, and adherent cell density estimated bycrystal violet staining and measuring A590. Slurp1 was found to inhibitMK/T-1 cells adhesion to collagen-I, -IV and fibronectin. The presenceof Slurp1 decreased the adhesion of MK/T-1 cells to three differentcomponents of the extracellular matrix tested.

FIG. 12. Effect of Slurp1 on MK/T-1 cell migration in in vitro gapfilling assays. MK/T-1 cells were infected with either 2× Tet-Off helperadenovirus (control), or 1× each of Slurp1-expressing adenovirus andTet-Off helper adenovirus (Slurp1). One day later, when the cells wereconfluent, a gap was generated by streaking with a 200 μl pipette tip.Gap filling by migration was monitored at 0 h, 18 h and 24 h post-gapgeneration. By 24 h, roughly 50% of the gap remains open in cellsinfected with Slurp1-expressing adenovirus while the gap in controlvirus infected cells is completely filled.

FIG. 13. Ligand-binding assay to identify Slurp1-interacting proteins.Kidney cells, which express Slurp1 and many uPAR ligands, and offer theadvantage of relatively abundant tissue availability, were used toscreen for Slurp1-interacting proteins. Kidney lysates were preparedusing either home-made Lysis Buffer (20 mM Tris-HCl (pH 8.0), 150 mMNaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.25% Tween-20) orM-PER (A non-denaturing commercial detergent formulation that extractssoluble protein). Kidney lysates were separated by non-denaturing PAGE,transferred to nitrocellulose membrane, blocked with 5% milk inphosphate buffered Tris saline (PBST), incubated with bovine serumalubumin (BSA) or partially purified His-Slurp1 (expressed in E. coli),and probed with anti-Slurp1 antibody to detect Slurp1-binding proteins(*). Blots were stripped and re-probed with anti-uPA antibody, andaligned with each other to detect Slurp1-interacting protein bandsoverlapping with those detected by anti-uPA antibody (**). There wereadditional Slurp1-interacting proteins (*).

FIGS. 14A-14B. ELISA Demonstrating Slurp1 interaction with uPA. ELISAplates were coated with partially purified maltose-binding protein (MBP)or MBP-Slurp1 fusion protein, and blocked with 5% milk. MBP-uPA fusionprotein (A), or cleaved uPA (B) was layered on coated MBP or MBP-Slurp1,washed, and Slurp1-bound MBP-uPA (A) or uPA (B) detected using anti-uPAantibody. MBP-Slurp1 efficiently interacted with both MBP-uPA fusionprotein (A) and cleaved, partially purified uPA (B).

FIG. 15. Pull-down assay Demonstrating Slurp1 interaction with uPA.Negative control MBP-Slurp1 (expressed in E. coli, purified throughamylose resin) or (ii) His-Slurp1 (expressed in E. coli) was bound onNi-ion resin, incubated with kidney lysate (as a source of uPA), washedthoroughly, bound proteins separated by SDS-PAGE and Slurp1-bound uPAdetected by anti-uPA antibody. uPA was detected only in His-Slurp1loaded columns, suggesting specific interaction between Slurp1 and uPA.

FIGS. 16A-16D. Slurp1 decreases the amount of cell surface bound uPA inquiescent and migrating MK/T-1 cells. MK/T-1 cells were plated oncollagen coated coverslip, surface bound uPA removed by acid wash (0.05Mglycine, pH-3.0) and neutralized by Hepes solution, and blocked withBSA. The cells were then treated with MBP or uPA, with or without Slurp1for 10 minutes, washed with PBS, and immunofluorescence was performedwith anti-uPA antibody. The number of foci on cell surface increasedwith uPA addition (panel B compared with panel A), and this increase wasnot seen when Slurp1 is added along with uPA (panel D compared withpanel C).

FIG. 17. Slurp1 decreases the amount of cell surface bound uPA inleading edges of migrating MK/T-1 cells. Following introduction oflinear gaps in confluent MK/T-1 cells with a 200 μl pipette tip, thecells were treated with Slurp1 or control protein for 4 hours, at theend of which uPA was detected in the leading edges by immunofluorescencewith rabbit anti-uPA antibody. While uPA was detected in the leadingedges of control protein treated migrating cells, it was markedlydecreased in those treated with Slurp1.

FIG. 18. Slurp1 expression is decreased in tears from inflamed humanocular surface. Tears were collected from male (M) or female (F) humanvolunteers with normal (N) or inflamed (I) ocular surface (cause ofinflammation was not distinguished) following informed consent. In asingle-blind experiment, equal amount of tear proteins were separated bySDS-PAGE, transferred to PVDF membrane and immunoblot performed withgoat anti-human SLURP1 antibody. While abundant SLURP1 expression wasdetected in tears from normal ocular surface, it was either decreased orcompletely absent in tears from inflamed human ocular surface,regardless of the gender.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [8123-90185-02_Sequence_Listing.txt, Nov. 4, 2013, 4.97 KB], whichis incorporated by reference herein. In the accompanying sequencelisting:

SEQ ID NO: 1 is an amino acid sequence of a human SLURP1 protein.

SEQ ID NO: 2 is an amino acid sequence of a mouse Slurp1 protein.

SEQ ID NO: 3 is an exemplary nucleic acid sequence encoding a humanSLURP1 protein.

SEQ ID NO: 4 is an exemplary nucleic acid sequence encoding a murineSlurp1 protein.

SEQ ID NOs: 5-6 are primer sequences.

SEQ ID NO: 7 is the nucleic acid sequence of the SLURP proximalpromoter.

DETAILED DESCRIPTION

It is disclosed herein that Slurp1 is a constitutively producedcomponent of corneal immune privilege that inhibits leukocyticinfiltration into the cornea in response to mild insults, and is rapidlydown-regulated when the cornea becomes infected, permitting protectiveinflammation to develop. Furthermore, Slurp1, and nucleic acids encodingSlurp1, can be used to treat ocular inflammation, such as, inflammationof an ocular surface. In specific non-limiting examples, theinflammation is corneal inflammation. In other embodiments the methodinhibits the migration of leukocytes into ocular surfaces, such as thecornea, conjunctiva or intraocular structures.

TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitatereview of the various embodiments of this disclosure, the followingexplanations of specific terms are provided.

Adenovirus: A virus of the family Adenoviridae, which are medium-sized(90-100 nm), nonenveloped icosahedral viruses composed of a nucleocapsidand a double-stranded linear DNA genome. The adenovirus genome islinear, non-segmented double-stranded (ds) DNA that is between 26 and 45kb. This allows the virus to theoretically carry 22 to 40 genes. Thelinear dsDNA genome is able to replicate in the nucleus of mammaliancells using the host's replication machinery. However, adenoviral DNAdoes not integrate into the genome and is not replicated during celldivision.

Adeno-associated Virus: Adeno-associated virus (AAV) is a small virusthat infects humans and some other primate species. AAV is not currentlyknown to cause disease and consequently the virus causes a very mildimmune response. AAV can infect both dividing and non-dividing cells andmay incorporate its genome into that of the host cell. The AAV genome isbuilt of single-stranded deoxyribonucleic acid (ssDNA), either positive-or negative-sensed, which is about 4.7 kilobase long. The genomecomprises inverted terminal repeats (ITRs) at both ends of the DNAstrand, and two open reading frames (ORFs): rep and cap. Rep is composedof four overlapping genes encoding Rep proteins required for the AAVlife cycle, and Cap contains overlapping nucleotide sequences of capsidproteins: VP1, VP2 and VP3, which interact together to form a capsid ofan icosahedral symmetry. For gene therapy, ITRs seem to be the onlysequences required in cis next to the therapeutic gene: structural (cap)and packaging (rep) genes can be delivered in trans.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Autoimmune disorder: A disorder in which the immune system produces animmune response (e.g., a B cell or a T cell response) against anendogenous antigen, with consequent injury to tissues. For example,rheumatoid arthritis is an autoimmune disorder, as are Hashimoto'sthyroiditis, pernicious anemia, Addison's disease, type I diabetes,Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, and Grave's disease,among others.

Blepharitis: Chronic inflammation of the eyelid. Signs and symptoms thatare associated with the chronic inflammation include redness of theeyelids, flaking of skin on the lids, crusting at the lid margins,generally worse on waking, cysts at the lid margin (hordeolum), redeyes, debris in the tear film, gritty sensation of the eye orforeign-body sensation, itching and eyes. The lids may become red andmay have ulcerative, non-healing areas which may actually bleed.Blepharitis does not tend to cause problems with the patient's visionwhatsoever, but due to a poor tear film, one may experience blurredvision.

Cornea: The transparent front part of the eye that covers the iris,pupil, and anterior chamber. Together with the lens, the cornea refractslight, and as a result helps the eye to focus, accounting forapproximately two-thirds of the eye's total optical power. The corneahas unmyelinated nerve endings sensitive to touch, temperature andchemicals; a touch of the cornea causes an involuntary reflex to closethe eyelid. The cornea does not have blood vessels; it receivesnutrients via diffusion from the tear fluid at the outside and theaqueous humor at the inside and also from neurotrophins supplied bynerve fibers that innervate it. In humans, the cornea has a diameter ofabout 11.5 mm and a thickness of 0.5-0.6 mm in the center and 0.6-0.8 mmat the periphery. The cornea has five layers; from the anterior toposterior these layers are the corneal epithelium, Bowman's layer, thecorneal stroma, Descemet's membrane, and the corneal endothelium.

Conjunctivitis: Inflammation of the conjunctiva, which lines the insideof the eyelids and covers the sclera. The conjunctiva is composed ofnon-keratinized stratified columnar epithelium with goblet cells. Thereare many types of conjunctivitis, including allergic conjunctivitis,bacterial conjunctivitis, viral conjunctivitis, and chemicalconjunctivitis. Generally in conjunctivitis the eye appears red, but thepupils are normally reactive to light and visual acuity is unchanged.

Non-limiting example of a conjunctivitis are viral conjunctivitis,bacterial conjunctivitis, fungal conjunctivitis, parasiticconjunctivitis, or allergic conjunctivitis. Acute conjunctivalinflammation is conjunctival inflammation that generally occurs for lessthan two weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12 or 13 days or less.Chronic conjunctival inflammation is conjunctival inflammation thatoccurs for at least two weeks such as for 3, 4, 5, 6, 7, 8, 9, 10 weeksor more, such as for months or years.

Conjunctivitis is characterized by presence or observation of two ormore (e.g., three, four, or five) of the following in a subject: anelevated number of T-lymphocytes (e.g., effector T-cells) in aconjunctiva, an elevated number of dendritic cells in a conjunctiva, anelevated number of macrophages in a conjunctiva, an elevated number ofstimulated monocytes in a conjunctiva, an elevated number of naturalkiller cells in a conjunctiva, an elevated number of B-cells in aconjunctiva, an elevated number of eosinophils in a conjunctiva, anelevated number of mast cells in a conjunctiva, an elevated level ofredness in a white of an eye or inner eyelid, pain in an eye,irritation, itchiness, burning, and/or dryness of an eye, excess tearsor other discharge from an eye, difficulty opening an eyelid, blurredvision, sensitivity to light, and swelling around an eye (e.g., ascompared to the levels in the same subject prior to conjunctivalinflammation, a subject not having an eye disorder (a healthy subject),or a threshold value).

The detection of an elevated level of the number of immunological cellspresent in the conjunctiva can be accomplished using methods known inthe art, such as in vivo confocal microscopy (see, e.g., Cruzat et al,Semin. Ophthalmol. 25: 171-177, 2010).

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of an antigenic epitope of Brachyury. Specific,non-limiting examples of a conservative substitution include thefollowing examples:

Original Residue Conservative Substitutions Al Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; LeuThe term conservative variant also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, and/or that the substituted polypeptideretains the function of the unsubstituted polypeptide. Non-conservativesubstitutions are those that reduce an activity or antigenicity.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. In anotherembodiment, a cytokine alters the maturation of lymphocytes, andinfluences isotype switching by B cells.

Immunosuppressive agent: A molecule, such as a chemical compound,polypeptide small molecule, steroid, nucleic acid molecule, or otherbiological agent, that can decrease an immune response such as aninflammatory reaction. Immunosuppressive agents include, but are notlimited to an agent of use in treating uveitis. Specific, non-limitingexamples of immunosuppressive agents are corticosteroids, cyclosporineA, FK506, and anti-CD4. In additional examples, the agent is abiological response modifier, such as KINERET® (anakinra), ENBREL®(etanercept), or REMICADE® (infliximab), a disease-modifyingantirheumatic drug (DMARD), such as ARAVA® (leflunomide). Agents of useto treat inflammation include non-steroidal anti-inflammatory drugs(NSAIDs), specifically a Cyclo-Oxygenase-2 (COX-2) inhibitor, such asCELEBREX® (celecoxib) and VIOXX® (rofecoxib), or another product, suchas HYALGAN® (hyaluronan) and SYNVISC® (hylan G-F20).

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Inflammation: A series of local tissue reactions that take place at asite of injury and have an immunological component. The injury may bedue to trauma, lack of blood supply, hemorrhage, autoimmune attack,transplanted exogenous tissue or infection. This generalized response bythe body includes the release of many components of the immune system(such as cytokines), attraction of cells to the site of the damage,swelling of tissue due to the release of fluid and other processes.Inflammation can be of an infectious or a non-infectious etiology. Inthe eye, inflammation produces vascular dilation, fluid leakage intoextra-vascular spaces, migration of leukocytes and other cells.

Infectious agent: An agent that can infect a subject, including, but notlimited to, viruses, bacteria and fungi.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids and proteins.

Keratitis: An inflammation or irritation of the cornea. Typical symptomsinclude red eye, foreign body sensation, pain, sensitivity to light,watery eyes, and blurred vision. Keratitis is the most common cause ofcorneal blindness caused by infection in the United States. It can becaused by injury to the cornea, dryness and/or inflammation of theocular surface, and infectious agents, such as herpes zoster and herpessimplex, and bacterial infections, such as Staphylococcus aureus andPseudomonas aeruginosa. There are other forms of keratitis, such asexposure keratitis, photokeratitis caused by exposure to ultravioletradiation, and allergic keratitis. Keratitis also can be caused byfungal infections (such as by Fusarium) and amoebic infections(Acanthamoeba). Infectious keratitis can progress rapidly, and generallyrequires urgent antibacterial, antifungal, or antiviral therapy toeliminate the pathogen. However, the underlying inflammation can causepersistent corneal injury (such as a scar) even after the infection orcorneal trauma has been successfully treated. Corticosteroids aresometimes used to treat such inflammation but they can have undesiredside effects such as increased intraocular pressure.

Superficial keratitis involves the superficial layers (the epithelium)of the cornea. Deep keratitis involves deeper layers of the cornea(including the epithelium, Bowman's Membrane and often the stroma).

Acute corneal inflammation is corneal inflammation that generally occursfor less than two weeks, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12 or13 days or less. Chronic corneal inflammation is corneal inflammationthat occurs for at least two weeks, such as for 3, 4, 5, 6, 7, 8, 9, 10weeks or more, such as for months or years.

In keratitis, the presence of two or more (e.g., three, four, or five)of the following is observed in a subject: an elevated number ofT-lymphocytes (e.g., effector T-cells) in a cornea, an elevated numberof dendritic cells in a cornea, an elevated number of macrophages in acornea, an elevated number of eosinophils in a cornea, an elevatednumber of mast cells in a cornea, an elevated number of B-cells in acornea, an elevated number of stimulated monocytes in a cornea, anelevated number of natural killer cells in a cornea, an elevated levelof redness in a cornea, pain in an eye, irritation, itchiness, burning,and/or dryness of a cornea, excess tears or other discharge from an eye,difficulty opening an eyelid, blurred vision, sensitivity to light, andswelling around the eye (e.g., as compared to the levels in the samesubject prior to corneal inflammation, a subject not having an eyedisorder (a healthy subject), or a threshold value). The detection of anelevated level of the number of immunological cells present in thecornea can be accomplished using methods known in the art, such as invivo confocal microscopy (see, e.g., Cruzat et al, Semin. Ophthalmol.25: 171-177, 2010). However, the existence of corneal inflammation alsocan be inferred from other underlying causes (such as trauma orinfection) or the appearance of the eye (such as redness and tearing).

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cells, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neutrophil: A type of phagocyte normally found in the blood. The nucleushas a characteristic lobed appearance, the separate lobes connected bychromatin. The nucleolus of a neutrophil disappears as the neutrophilmatures. Neutrophils quickly congregate at a focus of infection,attracted by cytokines expressed by activated endothelium, mast cells,and macrophages. Neutrophils express and release cytokines, which inturn amplify inflammatory reactions by several other cell types.Neutrophils are phagocytes. For targets to be recognized, they must becoated in opsonins. Each phagocytic event resulting in the formation ofa phagosome into which reactive oxygen species and hydrolytic enzymesare secreted. Neutrophils undergo a process called degranulation,wherein the contents of their granules can be released to combatinfection.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors including an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that includes therecombinant nucleic acid is referred to as a “recombinant host cell.”The gene is then expressed in the recombinant host cell to produce,e.g., a “recombinant polypeptide.” A recombinant nucleic acid may servea non-coding function (e.g., promoter, origin of replication,ribosome-binding site, etc.) as well.

A polynucleotide or nucleic acid sequence refers to a polymeric form ofnucleotide at least 10 bases in length. A recombinant polynucleotideincludes a polynucleotide that is not immediately contiguous with bothof the coding sequences with which it is immediately contiguous (one onthe 5′ end and one on the 3′ end) in the naturally occurring genome ofthe organism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms of DNA.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, intra-vitreously, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein are intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. Apolypeptide includes both naturally occurring proteins, as well as thosethat are recombinantly or synthetically produced. A polypeptide has anamino terminal (N-terminal) end and a carboxy-terminal end. In someembodiments, the polypeptide is a disclosed antibody or a fragmentthereof.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease such as an autoimmunedisorder. An example of a person with a known predisposition is someonewith a history of a disease in the family, or who has been exposed tofactors that predispose the subject to a condition. “Treatment” refersto a therapeutic intervention that ameliorates a sign or symptom of adisease or pathological condition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptide ornucleic acid preparation is one in which the peptide, protein or nucleicacid is more enriched than the peptide, protein or nucleic acid is inits natural environment within a cell. The term purified may be used torefer to both naturally occurring and recombinant molecules. Preferably,a preparation is purified such that the protein, peptide or nucleic acidrepresents at least 50% of the total peptide, protein or nucleic acidcontent of the preparation, such as is at least about 80%, 90%, 95%,98%, 99% or 100% of the content.

Replication defective: A viral vector that cannot further replicate andpackage its genomes. In one non-limiting example, when the cells of asubject are infected with a replication defective vector, a heterologousgene in the vector is expressed in the infected subject's cells.However, due to the fact that both the vector and the patient's cellslack essential genes for replication of the vector, it will not bepassed on to daughter cells when the infected cell divides. Examples ofessential genes for viral replication are the rev and cap genes for AAV,or gag, pol and env for a lentivirus. Generally, the genes necessary toreplicate and package are not present, such that and wild-type viruscannot be formed in the subject's cells.

Secreted Ly-6/uPAR-related protein 1 (SLURP1): A protein that in humansis encoded by the SLURP1 gene. The protein encoded by this gene is amember of the Ly6/uPAR family but lacks a GPI-anchoring signal sequence.Mutations in this gene have been associated with Mal de Meleda, a rareautosomal recessive skin disorder. This gene maps to the samechromosomal region as several members of the Ly6/uPAR family ofglycoprotein receptors. An exemplary human SLURP1 amino acid sequence isset forth as GENBANK Accession No. NP_(—)065160.1, incorporated hereinby reference. Additional exemplary SLURP1 amino acid sequences areprovided herein.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988, Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (ncbi.nlm.nihgov). The BLASTN program (for nucleotide sequences) uses as defaults aword length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5,N=−4, and a comparison of both strands. The BLASTP program (for aminoacid sequences) uses as defaults a word length (W) of 3, and expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915, 1989). An oligonucleotide is alinear polynucleotide sequence of up to about 100 nucleotide bases inlength.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. SLURP1, or a polynucleotideencoding SLURP1, are forms of therapeutic agents.

Therapeutically effective amount: A quantity of an agent sufficient toachieve a desired effect in a subject being treated. For instance, thiscan be the amount of a SLURP1 polypeptide, or a polynucleotide encodingthe SLURP1 polypeptide, sufficient to reduce inflammation, such as inthe cornea, or a dose sufficient to prevent advancement, or to causeregression of a disease, or which is capable of relieving symptomscaused by a disease, such as ocular inflammation. In one example, theamount is sufficient to prevent advancement, or to cause regression ofthe disease. In another example, the amount is sufficient to inhibit asign or symptom of inflammation, such as corneal inflammation, such asthe presence of inflammatory cells and/or redness and/or irritation thataccompanies the inflammation.

An effective amount of a SLURP1 polypeptide, or a polynucleotideencoding the SLURP1 polypeptide, can be administered systemically orlocally. In addition, an effective amount of a SLURP1 polypeptide, or apolynucleotide encoding the SLURP1 polypeptide, can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of the SLURP1 polypeptide, or apolynucleotide encoding the SLURP1 polypeptide, will be dependent on thepreparation applied, the subject being treated, the severity and type ofthe affliction, and the manner of administration of the compound.

The SLURP1 polypeptides, and polynucleotides encoding the SLURP1polypeptides, disclosed herein have equal applications in medical andveterinary settings. Therefore, the general terms “subject” and “subjectbeing treated” are understood to include all animals, including humansor other simians, dogs, cats, horses, and cows.

Uveal tract: The uveal tract is composed of three parts, the iris, theciliary body, and the choroid. It is the middle, vascular layer of theeye, protected externally by the cornea and the sclera. It contributesto the blood supply of the retina.

The iris is the anterior section of the ciliary body. It has arelatively flat surface with an aperture in the middle called the pupil.The iris lies in contact with the lens and divides the anterior chamberfrom the posterior chamber. The function of the iris is to control theamount of light that enters the eye.

The ciliary body extends forward from the anterior termination of thechoroid to the root of the iris. It is composed of two zones, the parsplicata and the pars plana. There are two layers of epithelium in theciliary body, the external pigmented and an internal non-pigmentedlayer. The ciliary body forms the root of the iris and governs the sizeof the lens. Aqueous humor is secreted by the ciliary processes into theposterior chamber of the eye.

The choroid is the posterior portion of the uveal tract and the middlepart of the eye, which lies between the retina and the sclera. It islargely composed of blood vessels. The function of the choroid is tonourish the outer portion of the underlying retina.

Uveitis: An intraocular inflammatory disease that includes iritis,cyclitis, panuveits, posterior uveitis and anterior uveitis. Iritis isinflammation of the iris. Cyclitis is inflammation of the ciliary body.Panuveitis refers to inflammation of the entire uveal (vascular) layerof the eye. Intermediate uveitis, also called peripheral uveitis, iscentered in the area immediately behind the iris and lens in the regionof the ciliary body and pars plana, and is also termed “cyclitis” and“pars planitis.”

“Posterior” uveitis generally refers to chorioretinitis (inflammation ofthe choroid and retina). Posterior uveitis can give rise to diversesymptoms but most commonly causes floaters and decreased vision similarto intermediate uveitis. Signs include cells in the vitreous humor,white or yellow-white lesions in the retina and/or underlying choroid,exudative retinal detachments, retinal vasculitis, and optic nerveedema.

Anterior uveitis refers to iridocyclitis (inflammation of the iris andthe ciliary body) and/or iritis. Anterior uveitis tends to be the mostsymptomatic, typically presenting with pain, redness, photophobia, anddecreased vision. Signs of anterior uveitis include pupillary miosis andinjections of the conjunctiva adjacent to the cornea, so-calledperilimbal flush. Biomicroscopic, or slit lamp, findings include cellsand flare in the aqueous humor as well as keratic precipitates, whichare clumps of cells and proteinaceous material adherent to the cornealendothelium. “Diffuse” uveitis implies inflammation involving all partsof the eye, including anterior, intermediate, and posterior structures.

“Acute” uveitis is a form of uveitis in which signs and symptoms occursuddenly and last for up to about six weeks. “Chronic” uveitis is a formin which onset is gradual and lasts longer than about six weeks.

The inflammatory products (i.e., cells, fibrin, excess proteins) ofocular inflammation are commonly found in the fluid spaces of the eye,i.e., anterior chamber, posterior chamber and vitreous space as well asinfiltrating the tissue imminently involved in the inflammatoryresponse.

Uveitis may occur following surgical or traumatic injury to the eye; asa component of an autoimmune disorder (such as rheumatoid arthritis,Bechet's disease, ankylosing spondylitis, sarcoidosis), as an isolatedimmune mediated ocular disorder (such as pars planitis oriridocyclitis), as a disease unassociated with known etiologies, andfollowing certain systemic diseases which cause antibody-antigencomplexes to be deposited in the uveal tissues. Uveitis includes ocularinflammation associated with Bechet's disease, sarcoidosis,Vogt-Koyanagi-Harada syndrome, birdshot chorioretinopathy andsympathetic ophthalmia. Thus, non-infectious uveitis occurs in theabsence of an infectious agent.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

SLURP1 Polypeptides, Polynucleotides Encoding SLURP1 and PharmaceuticalCompositions for Treatment of Ocular Inflammation

Methods are disclosed herein for treating ocular inflammation. Thesemethods disclosed herein utilize SLURP1 polypeptides and/or nucleicacids that encode SLURP1 polypeptides. An exemplary human SLURP1 proteinis:

-   -   MASRWAVQLL LVAAWSMGCG EALKCYTCKE PMTSASCRTI TRCKPEDTAC        MTTLVTVEAEYPFNQSPVVT RSCSSSCVAT DPDSIGAAHL IFCCFRDLCN SEL (SEQ        ID NO: 1, see GENBANK Accession No. NP_(—)065160.1, Jun. 29,        2012, incorporated herein by reference).

An exemplary murine Slurp1 protein is

-   -   MTLRWAMWLLLLAAWSMGYGEAFRCYTCEQPTAINSCKNIAQCKM        EDTACKTVLETVEAAFPFNHSPMVTRSCSSSCLATDPDGIGVAHPVF CCFRDLCNSG (SEQ        ID NO: 2, see GENBANK Accession No. NM_(—)020519.1, Jun. 29,        2012, incorporated herein by reference).        In some embodiments, the SLURP1 polypeptide comprises or        consists of amino acids 23-103 of the amino acid sequence set        forth as SEQ ID NO: 1 or SEQ ID NO: 2. Homologs and variants,        such as polypeptides about 95%, 96%, 97%, 98%, 99% identical to        these polypeptides are also of use in the methods disclosed        herein. In some embodiments, polypeptides 95% identical to the        amino acids set forth as residues 23-103 of SEQ ID NO: 1, such        as at least 95%, at least 96%, at least 97%, at least 98%, at        least 99%, or 100% identical to the amino acids set forth as        residues 23-103 of SEQ ID NO: 1 are of use in the methods        disclosed herein. In further embodiments, the polypeptide        includes at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 conservative        substitutions in SEQ ID NO: 1, wherein the polypeptide retains        an anti-inflammatory activity, such as reducing neutrophil        infiltration in an ocular surface.

An exemplary nucleic acid encoding a human SLURP1 polypeptide is

-   -   CTCTCATCAC TTCTGAGCAC GGAGCAATGG CCTCTCGCTG GGCTGTGCAG        CTGCTGCTCGTGGCAGCCTG GAGCATGGGC TGTGGTGAGG CCCTCAAGTG CTACACCTGC        AAGGAGCCCATGACCAGTGC TTCCTGCAGG ACCATTACCC GCTGCAAGCC AGAGGACACA        GCCTGCATGACCACGCTGGT GACGGTGGAG GCAGAGTACC CCTTCAACCA GAGCCCCGTG        GTGACCCGCTCCTGCTCCAG CTCCTGTGTG GCCACCGACC CCGACAGCAT CGGGGCCGCC        CACCTGATCTTCTGCTGCTT CCGAGACCTC TGCAACTCGG AACTCTGAAC CCAGGGCGGC        AGGGCGGAAGGTGCTCCTCA GGCACCTCCT CTCTGACGGG GCCTGGCTCC ACCTGTGATC        ACCTCCCCCTGCTTCCTGCT GCTGTGGCAC AGCTCACTCA TGGGGTCTGA GGGGAGAGAA        GCACACCAGGGGCGCCCTCT GCCTTCCATA CCCCACGCTT ATAAAACATA ACTAAGCCAA        (SEQ ID NO: 3, see GENBANK Accession No. NM_(—)020427.2, Jun.        29, 2012, incorporated herein by reference).        An exemplary nucleic acid encoding mouse Slurp1 is:    -   AGGGCTCCTA GCTCCTGAGC ACTGAAGAAT GACCCTTCGC TGGGCCATGT        GGCTGCTGCT CTTGGCAGCC TGGAGCATGG GCTATGGTGA GGCCTTCCGA        TGCTATACCT GTGAGCAGCC CACGGCCATT AACTCATGCA AGAATATTGC        TCAGTGCAAG ATGGAAGACA CAGCCTGTAAGACTGTACTG GAGACAGTGG AAGCAGCGTT        CCCCTTCAAC CACAGTCCCA TGGTGACCCG CTCCTGCTCC AGCTCGTGTC        TGGCCACCGA CCCTGATGGC ATTGGCGTTG CCCATCCTGT CTTCTGTTGC        TTCCGTGACC TCTGCAACTC AGGGTTTCCA GGCTTCGTGG CAGGCCTCTA        GCCACACAGG GAGCCTCCTC GTTCCTTCTC TATCCACTCT CCCGGCAGGG        CCCGGTGCTG CCTGCAGTCG TCTCTACATG CCTGGATCTA TGAGCAGAGC        TCACTGAGCC TCAGGTCACT CACTGTCCAC CAAGCTTGTG GAAAATAAAA        TAAACCAAGG GCGAA (SEQ ID NO: 4, see GENBANK Accession No.        NM_(—)020519.12, Apr. 18, 2013, incorporated herein by        reference).

Slurp1 polypeptides and polynucleotides are disclosed in U.S. Pat. No.7,960,398 and U.S. Pat. No. 7,691,808 which are both incorporated byreference herein.

In some embodiments, the nucleic acid molecules include a nucleic acidsequence encoding an amino acid sequence at least 95% identical to theamino acids set forth as residues 23-103 of SEQ ID NO: 1, such as atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acids set forth as residues 23-103 of SEQ IDNO: 1. In additional embodiments, the nucleic acid molecules include anucleic acid sequence encoding an amino acid sequence at least 95%identical to the amino acids set forth as SEQ ID NO: 1, such as at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to the amino acids set forth as of SEQ ID NO: 1. In furtherembodiments, the nucleic acid encodes a polypeptide includes at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39or 40 conservative substitutions in SEQ ID NO: 1.

These polynucleotides include DNA, cDNA and RNA sequences which encodethe polypeptide of interest. Silent mutations in the coding sequenceresult from the degeneracy (i.e., redundancy) of the genetic code,whereby more than one codon can encode the same amino acid residue.Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, orTTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC;asparagine can be encoded by AAT or AAC; aspartic acid can be encoded byGAT or GAC; cysteine can be encoded by TGT or TGC; alanine can beencoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA orCAG; tyrosine can be encoded by TAT or TAC; and isoleucine can beencoded by ATT, ATC, or ATA. Tables showing the standard genetic codecan be found in various sources (e.g., L. Stryer, 1988, Biochemistry,3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

Nucleic acid molecules encoding SLURP1 can readily be produced by one ofskill in the art, using the amino acid sequences provided herein, andthe genetic code. Nucleic acid sequences encoding SLURP1 can be preparedby any suitable method including, for example, cloning of appropriatesequences or by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979;the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151,1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence, or by polymerization with a DNA polymerase using the singlestrand as a template. Exemplary nucleic acids including sequencesencoding SLURP1 can be prepared by cloning techniques.

A nucleic acid encoding a SLURP1 polypeptide can be cloned or amplifiedby in vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA using primers based on the DNA sequence of themolecule. A wide variety of cloning and in vitro amplificationmethodologies are well known to persons skilled in the art. PCR methodsare described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989). Polynucleotides also can beisolated by screening genomic or cDNA libraries with probes selectedfrom the sequences of the desired polynucleotide under stringenthybridization conditions.

In the context of the compositions and methods described herein, anucleic acid sequence that encodes a SLURP1 polypeptide, such asdescribed above, is incorporated into a vector capable of expression ina host cell, using established molecular biology procedures. For examplenucleic acids, such as cDNAs, that encode SLURP1 can be manipulated withstandard procedures such as restriction enzyme digestion, fill-in withDNA polymerase, deletion by exonuclease, extension by terminaldeoxynucleotide transferase, ligation of synthetic or cloned DNAsequences, site-directed sequence-alteration via single-strandedbacteriophage intermediate or with the use of specific oligonucleotidesin combination with PCR or other in vitro amplification.

Exemplary procedures sufficient to guide one of ordinary skill in theart through the production of vector capable of expression in a hostcell (such as an adenoviral vector) that includes a polynucleotidesequence that encodes a SLURP1 polypeptide can be found for example inSambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Sambrook et al., MolecularCloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001;Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates, 1992 (and Supplements to 2003); and Ausubel etal., Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999.

Typically, a polynucleotide sequence encoding a SLURP1 polypeptide isoperably linked to transcriptional control sequences including, forexample a promoter and a polyadenylation signal. A promoter is apolynucleotide sequence recognized by the transcriptional machinery ofthe host cell (or introduced synthetic machinery) that is involved inthe initiation of transcription. A polyadenylation signal is apolynucleotide sequence that directs the addition of a series ofnucleotides on the end of the mRNA transcript for proper processing andtrafficking of the transcript out of the nucleus into the cytoplasm fortranslation.

Exemplary promoters include viral promoters, such as cytomegalovirusimmediate early gene promoter (“CMV”), herpes simplex virus thymidinekinase (“tk”), SV40 early transcription unit, polyoma, retroviruses,papilloma virus, hepatitis B virus, and human and simianimmunodeficiency viruses. Other promoters are isolated from mammaliangenes, including the immunoglobulin heavy chain, immunoglobulin lightchain, T-cell receptor, HLA DQ α and DQ β, β-interferon, interleukin-2,interleukin-2 receptor, MHC class II, HLA-DRα, β-actin, muscle creatinekinase, prealbumin (transthyretin), elastase I, metallothionein,collagenase, albumin, fetoprotein, β-globin, c-fos, c-HA-ras, insulin,neural cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B)histone, type I collagen, glucose-regulated proteins (GRP94 and GRP78),rat growth hormone, human serum amyloid A (SAA), troponin I (TNI),platelet-derived growth factor, and dystrophin, and promoters specificfor keratinocytes, and epithelial cells.

The promoter can be either inducible or constitutive. An induciblepromoter is a promoter which is inactive or exhibits low activity exceptin the presence of an inducer substance. Examples of inducible promotersinclude, but are not limited to, MT II, MMTV, collagenase, stromelysin,SV40, murine MX gene, α-2-macroglobulin, MHC class I gene h-2 kb, HSP70,proliferin, tumor necrosis factor, or thyroid stimulating hormone genepromoter.

Typically, the promoter is a constitutive promoter that results in highlevels of transcription upon introduction into a host cell in theabsence of additional factors. Optionally, the transcription controlsequences include one or more enhancer elements, which are bindingrecognition sites for one or more transcription factors that increasetranscription above that observed for the minimal promoter alone.

It may be desirable to include a polyadenylation signal to effect propertermination and polyadenylation of the gene transcript. Exemplarypolyadenylation signals have been isolated from bovine growth hormone,SV40 and the herpes simplex virus thymidine kinase genes. Any of theseor other polyadenylation signals can be utilized in the context of theadenovirus vectors described herein.

The polynucleotides encoding a SLURP1 polypeptide include a recombinantDNA which is incorporated into a vector in an autonomously replicatingplasmid or virus or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (such as a cDNA) independent ofother sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

Viral vectors can also be prepared encoding the SLURP1 polypeptides. Anumber of viral vectors have been constructed, including polyoma, SV40(Madzak et al., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner,1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988,Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol.,66:4407-4412; Quantin et al., 1992, Proc. Nad. Acad. Sci. USA,89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson etal., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al.,1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al., 1992,Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992,Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene,89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992, CumTop. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell. Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell. Biol., 5:431-437; Sorge etal., 1984, Mol. Cell. Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

Thus, in one embodiment, the polynucleotide encoding a SLURP1polypeptide is included in a viral vector. Suitable vectors includeretrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors,capripox vectors, suipox vectors, adenoviral vectors, herpes virusvectors, alpha virus vectors, baculovirus vectors, Sindbis virusvectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus, yeast and the like.

Adenovirus vectors (Ad) vectors can be produced that encode a SLURP1polypeptide and are of use in the methods disclosed herein. Thesevectors are of use in the methods disclosed herein, includingreplication competent, replication deficient, gutless forms thereof, andadeno-associated virus (AAV) vectors. Without being bound by theory,adenovirus vectors are known to exhibit strong expression in vitro,excellent titer, and the ability to transduce dividing and non-dividingcells in vivo (Hitt et al., Adv in Virus Res 55:479-505, 2000). Whenused in vivo these vectors lead to strong but transient gene expressiondue to immune responses elicited to the vector backbone.

Adenoviral vectors are often constructed by insertion of a nucleic acidencoding a SLURP1 polypeptide in place of, or in the middle of,essential viral sequences such as those found at the E1 region ofadenovirus (Berkner, BioTechniques, 6:616-629, 1988; Graham et al.,Methods in Molecular Biology, 7:109-128, Ed: Murcy, The Human PressInc., 1991). Inactivation of essential viral genes by, for example,deletion or insertion, disables the adenovirus' ability to replicate. Topropagate such vectors in cell culture, the deleted genes must beprovided in trans (for example, the E1A and E1B proteins in the case ofan E1 delete vector). These replication-defective adenoviruses areproduced in packaging cells engineered to complement thereplication-incompetent virus by expressing the subset of geneticelements deleted from their viral genome. Potential sites for theinsertion of a nucleic acid of interest, such as a nucleic acid encodinga SLURP1 polypeptide, in recombinant adenoviral vectors include, withoutlimitation, the E1, E2, E3 and the E4 region. In some embodiments, arecombinant adenoviral vector is produced from a human adenovirus thathas the E1 region deleted and replaced with a nucleic acid encoding aSLURP1 polypeptide. The resulting viral vector, with one or more of itsessential genes inactivated, is replication defective(Statford-Perricaudet et al., Human Gene Therapy, 1:241-256, 1990).

The recombinant adenovirus vectors can include: (1) a packaging siteenabling the vector to be incorporated into replication-defective Advirions; and (2) the nucleic acid encoding the SLURP1 polypeptide. Otherelements of use for incorporation into infectious virions, include the5′ and 3′ Ad ITRs; the E2 and E3 genes can be included in the vector. Insome embodiments, a nucleic acid encoding a SLURP1 polypeptide isinserted into adenovirus in the deleted E1A, E1B or E3 region of thevirus genome. In some embodiments, the adenovirus vectors do not expressone or more wild-type adenovirus gene products, such as E1a, E1b, E2,E3, E4. In some non-limiting examples, virions are typically usedtogether with packaging cell lines that complement the functions of E1,E2A, E4 and optionally the E3 gene regions (see, for example, U.S. Pat.Nos. 5,872,005, 5,994,106, 6,133,028 and 6,127,175, incorporated byreference herein in their entirety). Adenovirus vectors can be purifiedand formulated using techniques known in the art.

In some embodiments, packaging cell lines such as the human embryonickidney 293 (“HEK-293” or “293”) cell line (Graham et al., J. Gen.Virol., 36:59-72, 1977) or human embryonic retinoblast (“HER-911” or“911”) cell line (Fallaux et al., Hum. Gene Ther., 7:215-222, 1996),provide in trans the missing region, such as the E1 region, so that thedeleted or modified adenoviral vector can replicate in such cells.Suitable adenoviral vectors are disclosed, for example, in U.S. PatentPublication No. 20080193484, which is incorporated herein by reference.Replication-defective adenovirus virions encapsulating the recombinantadenovirus vectors can be made by standard techniques known in the artusing packaging cells and packaging technology. Examples of thesemethods can be found, for example, in U.S. Pat. No. 5,872,005,incorporated herein by reference in its entirety.

Recombinant AAV vectors are characterized in that they are capable ofdirecting the expression and the production of the selected transgenicproducts in targeted cells. Thus, the recombinant vectors comprise atleast all of the sequences of AAV essential for encapsidation and thephysical structures for infection of target cells.

Recombinant AAV (rAAV) virions can be constructed such that theyinclude, as operatively linked components in the direction oftranscription, control sequences including transcriptional initiationand termination sequences, and the nucleic acid encoding the SLURP1polypeptide. These components are bounded on the 5′ and 3′ end byfunctional AAV inverted terminal repeat (ITR) sequences. By “functionalAAV ITR sequences” is meant that the ITR sequences function as intendedfor the rescue, replication and packaging of the AAV virion. Hence, AAVITRs for use in the vectors need not have a wild-type nucleotidesequence, and can be altered by the insertion, deletion or substitutionof nucleotides, or the AAV ITRs can be derived from any of several AAVserotypes, provided they are functional. An AAV vector is a vectorderived from an adeno-associated virus serotype, including withoutlimitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, etc.In some embodiments, the AAV vectors have the wild type REP and CAPgenes deleted in whole or part, but retain functional flanking ITRsequences. These vectors can all be used, without limitation, for theexpression of a Slurp 1 polypeptide.

It is understood that portions of the nucleic acid sequences encodingSLURP1 polypeptides can be deleted as long as the polypeptides arefunctionally active. For example, it may be desirable to delete one ormore amino acids from the N-terminus, C-terminus, or both. It is alsocontemplated that the substitution of residues in the disclosed SLURP1polypeptides can be made, for example conservative substitutions, suchthat the ability of the functionality of the SLURP1 polypeptides ismaintained.

The SLURP1 polypeptide, or a polynucleotide encoding the SLURP1polypeptide, described herein can be formulated in a variety of waysdepending on the location and type of disease to be treated.Pharmaceutical compositions are thus provided for both local use (forexample, topical or within an ocular transplant), as well as forsystemic use. The subject can be any subject, such as a mammaliansubject. Therefore, the disclosure includes within its scopepharmaceutical compositions comprising at least one SLURP1 polypeptide,or a polynucleotide encoding the SLURP1 polypeptide, formulated for usein human or veterinary medicine. Any of these compositions are of use inthe methods disclosed herein.

The SLURP1 polypeptides and nucleic acids encoding SLURP1 polypeptidescan be administered ex vivo (such as into a stem cell to be implantedinto the eye) or in vivo to a cell or subject. Generally, it isdesirable to prepare the compositions as pharmaceutical compositionsappropriate for the intended application. Accordingly, methods formaking a medicament or pharmaceutical composition containing thepolypeptides, nucleic acids, or vectors described above are includedherein. Typically, preparation of a pharmaceutical composition(medicament) entails preparing a pharmaceutical composition that isessentially free of pyrogens, as well as any other impurities that couldbe harmful to humans or animals. Typically, the pharmaceuticalcomposition contains appropriate salts and buffers to render thecomponents of the composition stable and allow for uptake of nucleicacids or virus by target cells.

Therapeutic compositions can be provided as parenteral compositions,such as for injection or infusion. Such compositions are formulatedgenerally by mixing a disclosed therapeutic agent at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, for example onethat is non-toxic to recipients at the dosages and concentrationsemployed and is compatible with other ingredients of the formulation. Inaddition, a disclosed therapeutic agent can be suspended in an aqueouscarrier, for example, in an isotonic buffer solution at a pH of about3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citricacid and sodium phosphate-phosphoric acid, and sodium acetate/aceticacid buffers. The active ingredient, optionally together withexcipients, can also be in the form of a lyophilisate and can be madeinto a solution prior to parenteral administration by the addition ofsuitable solvents. Solutions such as those that are used, for example,for parenteral administration can also be used as infusion solutions.

Pharmaceutical compositions can include an effective amount of thepolypeptide, nucleic acid, or dispersed (for example, dissolved orsuspended) in a pharmaceutically acceptable carrier or excipient.Pharmaceutically acceptable carriers and/or pharmaceutically acceptableexcipients are known in the art and are described, for example, inRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995).

The nature of the carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition, pharmaceutical compositions to be administeredcan contain minor amounts of non-toxic auxiliary substances, such aswetting or emulsifying agents, preservatives, and pH buffering agentsand the like, for example sodium acetate or sorbitan monolaurate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. For example, certainpharmaceutical compositions can include the vectors or viruses in water,mixed with a suitable surfactant, such as hydroxypropylcellulose.Dispersions also can be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

Administration of therapeutic compositions can be by any common route aslong as the target tissue (typically, the ocular surface) is availablevia that route. This includes oral, nasal, ocular, buccal, or othermucosal (such as rectal or vaginal) or topical administration.Alternatively, administration will be by orthotopic, intradermalsubcutaneous, intramuscular, intraperitoneal, or intravenous injectionroutes. In some embodiments, the SLURP1 polypeptide or thepolynucleotide encoding the SLURP1 polypeptide is formulated foradministration to the eye, such as to the cornea, uveal tract, orconjunctiva. Such pharmaceutical compositions are usually administeredas pharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients. Pharmaceuticalcompositions that include SLURP1, and/or a polynucleotide encodingSLURP1, as an active ingredient, can be formulated with an appropriatesolid or liquid carrier, depending upon the particular mode ofadministration chosen.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical and oral formulations can be employed.Topical preparations can include eye drops, ointments, sprays and thelike. Eye drops or sprays can be provided in unit dose dispensers (suchas eye drop bottles that dispense a metered unit dose that contains theSLURP1 polypeptide, or polynucleotide encoding the SLURP1 polypeptide,either alone or in combination with other therapeutic agents such ascorticosteroids). Oral formulations may be liquid (e.g., syrups,solutions, or suspensions), or solid (e.g., powders, pills, tablets, orcapsules). For solid compositions, conventional non-toxic solid carrierscan include pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. Actual methods of preparing such dosage forms areknown, or will be apparent, to those of ordinary skill in the art.Implants can also be employed (see below).

The pharmaceutical compositions that include a SLURP1 polypeptide, or anucleic acid encoding the SLURP1 polypeptide, in some embodiments, willbe formulated in unit dosage form, suitable for individualadministration of precise dosages. The amount of active compound(s)administered will be dependent on the subject being treated, theseverity of the affliction, and the manner of administration, and isbest left to the judgment of the prescribing clinician. Within thesebounds, the formulation to be administered will contain a quantity ofthe active component(s) in amounts effective to achieve the desiredeffect in the subject being treated.

The SLURP1 polypeptide, or a nucleic acid encoding the SLURP1polypeptide can be included in an inert matrix for either topicalapplication or injection into the eye, such as for intra-vitrealadministration. As one example of an inert matrix, liposomes may beprepared from dipalmitoyl phosphatidylcholine (DPPC), such as eggphosphatidylcholine (PC). Liposomes, including cationic and anionicliposomes, can be made using standard procedures as known to one skilledin the art. Liposomes including a SLURP1 polypeptide, or a nucleic acidencoding the SLURP1 polypeptide can be applied topically, either in theform of drops or as an aqueous based cream, or can be injectedintraocularly. In a formulation for topical application, the SLURP1polypeptide, or a nucleic acid encoding the SLURP1 polypeptide is slowlyreleased over time as the liposome capsule degrades due to wear and tearfrom the eye surface. In a formulation for intraocular injection, theliposome capsule degrades due to cellular digestion. Both of theseformulations provide advantages of a slow release drug delivery system,allowing the subject to be exposed to a substantially constantconcentration of the SLURP1 polypeptide, or a nucleic acid encoding theSLURP1 polypeptide, over time. In one example, the SLURP1 polypeptide,or a nucleic acid encoding the SLURP1 polypeptide, can be dissolved inan organic solvent such as DMSO or alcohol as previously described andcontain a polyanhydride, poly(glycolic) acid, poly(lactic) acid, orpolycaprolactone polymer.

The SLURP1 polypeptide, or a nucleic acid encoding the SLURP1polypeptide, can be included in a delivery system that can be implantedat various sites in the eye, depending on the size, shape andformulation of the implant, and the type of transplant procedure. TheSLURP1 polypeptide, or a nucleic acid encoding the SLURP1 polypeptide,can be used alone. However, in another embodiment, at least oneadditional agent, such as at least one agent that is disclosed below,can be included along with the SLURP1 polypeptide, or a nucleic acidencoding the SLURP1 polypeptide, in the delivery system, such as in animplant. The delivery system is then introduced into the eye. Suitablesites include but are not limited to the anterior chamber, anteriorsegment, posterior chamber, posterior segment, vitreous cavity,suprachoroidal space, subconjunctiva, episcleral, intracorneal,epicorneal and sclera. In one example, the delivery system is placed inthe anterior chamber of the eye. In another example, the delivery systemis placed in the vitreous cavity.

The implants can be inserted into the eye by a variety of methods,including placement by forceps or by trocar following making an incisionin the sclera (for example, a 2-3 mm incision) or other suitable site.In some cases, the implant can be placed by trocar without making aseparate incision, but instead by forming a hole directly into the eyewith the trocar. The method of placement can influence the releasekinetics. For example, implanting the device into the vitreous or theposterior chamber with a trocar may result in placement of the devicedeeper within the vitreous than placement by forceps, which may resultin the implant being closer to the edge of the vitreous. The location ofthe implanted device may influence the concentration gradients of SLURP1polypeptide, or a nucleic acid encoding the SLURP1 polypeptide,surrounding the device, and thus influence the release rates (forexample, a device placed closer to the edge of the vitreous may resultin a slower release rate, see U.S. Pat. No. 5,869,079 and U.S. Pat. No.6,699,493).

The use of implants is well known in the art (see U.S. Pat. No.6,699,493 and U.S. Pat. No. 5,869,079). In one embodiment, an implant isformulated with the SLURP1 polypeptide, or a nucleic acid encoding theSLURP1 polypeptide, associated with a bioerodible polymer matrix.

Generally, when implants are used, the SLURP1 polypeptide, or nucleicacid encoding the SLURP1 polypeptide, is homogeneously distributedthrough the polymeric matrix, such that it is distributed evenly enoughthat no detrimental fluctuations in rate of release occur because ofuneven distribution of the immunosuppressive agent in the polymermatrix. The selection of the polymeric composition to be employed varieswith the desired release kinetics, the location of the implant, patienttolerance, and the nature of the implant procedure. The polymer can beincluded as at least about 10 weight percent of the implant. In oneexample, the polymer is included as at least about 20 weight percent ofthe implant. In another embodiment, the implant comprises more than onepolymer. These factors are described in detail in U.S. Pat. No.6,699,493. Characteristics of the polymers generally includebiodegradability at the site of implantation, compatibility with theagent of interest, ease of encapsulation, and water insolubility,amongst others. Generally, the polymeric matrix is not fully degradeduntil the drug load has been released. The chemical composition ofsuitable polymers is known in the art (for example, see U.S. Pat. No.6,699,493).

The SLURP1 polypeptides and polynucleotides disclosed herein can beformulated in an implantable form with other carriers and solvents. Forexample, buffering agents and preservatives can be employed. Watersoluble preservatives include sodium bisulfite, sodium bisulfate, sodiumthiosulfate, benzalkonium chloride, chlorobutanol, thimerosal,phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinylalcohol and phenylethyl alcohol. These agents can be present inindividual amounts of from about 0.001 to about 5% by weight, such asabout 0.01 to about 2%. Suitable water soluble buffering agents that maybe employed are sodium carbonate, sodium borate, sodium phosphate,sodium acetate, sodium bicarbonate. These agents can be present inamounts sufficient to maintain a pH of the system of between about 2 toabout 9 such as about 4 to about 8, or at about 6 to about 7. In oneexample, the pH of the system is maintained at about 7. As such, thebuffering agent can be as much as 5% on a weight-to-weight basis of thetotal composition. Electrolytes such as sodium chloride and potassiumchloride may also be included in the formulation. The proportions ofsuppressive ODN, polymer, and any other modifiers may be empiricallydetermined by formulating several implants with varying proportions. AUSP approved method for dissolution or release test can be used tomeasure the rate of release (USP 23; NF 18 (1995) pp. 1790-1798). Theimplant sizes and shape can also be varied for use in particular regionsof the eye (see U.S. Pat. No. 5,869,079).

The SLURP1 polypeptides, and polynucleotide encoding these polypeptides,can be included in a contact lens, such as a bandage lens, for treatingcorneal inflammation in a subject. The contact lens includes a contactlens substrate and a coating provided on at least a portion of thesubstrate, or within the matrix of the lens material. The coating caninclude an amount of SLURP1 polypeptide effective to treat cornealinflammation in a subject upon administration of the contact lens to thesubject. Coatings including the therapeutic agent can be applied to anumber of contact lens substrate materials known in the art. Virtuallyany substrate known in the art that can be fashioned into a contact lenscan be used provided it is optically transparent.

In some embodiments, the substrate can include optically transparentmaterials that allow oxygen to reach the cornea in an amount, which issufficient for long-term corneal health. Examples of substrates includepolymers made from hydrophobic materials, such as silicone copolymers,interpolymers, oligomers, and macromers. Illustrative polysilicones arepolydimethyl siloxane, polydimethyl-co-vinylmethylsiloxane. Othersilicones include silicone rubbers described in U.S. Pat. No. 3,228,741;U.S. Pat. No. 3,341,490; U.S. Pat. No. 3,518,324. Substrates describedin U.S. Pat. No. 4,136,250; U.S. Pat. No. 5,387,623; U.S. Pat. No.5,760,100; U.S. Pat. No. 5,789,461; U.S. Pat. No. 5,776,999; U.S. Pat.No. 5,849,811; U.S. Pat. No. 5,314,960 and U.S. Pat. No. 5,244,981 canalso be used. Cross-linked polymers of propoxylate of methyl glucose andpropylene oxide and HEMA-based hydrogels can also be used as substratesof the contact lens.

Silicone compositions that can be used in forming the contact lens arethe cross-linked polysiloxanes obtained by cross-linking siloxaneprepolymers by means of hydrosilylation, co-condensation and by freeradical mechanisms as in U.S. Pat. No. 4,143,949. Additionalsilicone-based substrates are cross-linked polymers of αomega.-bisamionpropyl polydimethylsiloxane, and gylycidyl methacrylate,cross-linked polymers. Silicone compositions can also be made fromcombining a methacrylate with one or more silicone monomers in thepresence of a group transfer polymerization (GTP) catalyst to form amacromer that is subsequently polymerized with other monomers to givethe final substrate. Substrates described in U.S. Pat. No. 6,951,894 arealso suitable for use in the present invention.

The therapeutic agent can be prepared and applied to the contact lens asan aqueous solution, suspension, or colloid and then applied to thecontact lens substrate according to any process that can provide thecoating in contact with the substrate or cause the therapeutic agent tobe absorbed within the contact lens. For example, processes for applyingthe coating to the substrate include immersion, spraying, brushing, andspin coating. Once the lens substrate is coated it may be subjected toany number of additional steps that are conducted in the manufacture ofcontact lenses. These can include, for example, swelling and washingsteps, the addition of additives such as surfactants, and extractionsteps, amongst others. The solution including the SLURP1 polypeptide, ora nucleic acid encoding this polypeptide, can adhere to the contact lensby, for example, chemical bonding, such as covalent or ionic bonding, orphysical bonding. In some aspects, the coating is released from the lenssubstrate throughout its useful life (such as storage time plus the timein which it will be in contact with a user's eye).

The contact lens can also include more than one layer of coating. Thiscan be desirable where the coating layer will provide the requisitesurface properties (e.g. treatment of corneal inflammation) but is notparticularly compatible with the lens substrate itself. For example, atie-layer or coupling agent can be used to adhere the coating to thesubstrate. Selections of compatible lens substrate, therapeutic coating,and tie-layer (if necessary) materials is well within the knowledge ofone skilled in the art. Generally, the contact lens is non-toxic to thesubject's cornea and other tissue while providing for the treatment ofcorneal inflammation in the subject.

The SLURP1 polypeptide, and/or a polynucleotide encoding the SLURP1polypeptide described herein can be formulated with other carriers andsolvents. For example, buffering agents and preservatives can beemployed. Water soluble preservatives include sodium bisulfite, sodiumbisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate,methylparaben, polyvinyl alcohol and phenylethyl alcohol. These agentscan be present in individual amounts of from about 0.001 to about 5% byweight, such as about 0.01 to about 2%. Suitable water soluble bufferingagents that may be employed are sodium carbonate, sodium borate, sodiumphosphate, sodium acetate, sodium bicarbonate. These agents can bepresent in amounts sufficient to maintain a pH of the system of betweenabout 2 to about 9 such as about 4 to about 8, or at about 6 to about 7.In one example, the pH of the system is maintained at about 7. As such,the buffering agent can be as much as 5% on a weight-to-weight basis ofthe total composition. Electrolytes such as sodium chloride andpotassium chloride may also be included in the formulation. Theproportions of SLURP1 polypeptide, and/or a polynucleotide encoding theSLURP1 polypeptide, polymer, and any other modifiers may be empiricallydetermined by formulating several implants with varying proportions. AUSP approved method for dissolution or release test can be used tomeasure the rate of release (USP 23; NF 18 (1995) pp. 1790-1798).Implant sizes and shape can also be varied for use in particular regionsof the eye (see U.S. Pat. No. 5,869,079).

The SLURP1 polypeptide, and/or a polynucleotide encoding the SLURP1polypeptide can be formulated with additional therapeutic agents.Exemplary agents include cyclosporine, FK506, steroids such ashydrocortisone, antibodies (such as anti-CD4 or antibodies thatspecifically bind the IL-2 receptor), cytokines (such asbeta-interferon), or non-steroidal anti-inflammatory agents. Additionalagents include antibiotics, such as minoglycosides (for example,amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin,dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin, kanamycin,micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin,ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin,trospectomycin), amphenicols (for example, azidamfenicol,chloramphenicol, florfenicol, thiamphenicol), ansamycins (for example,rifamide, rifampin, rifamycin sv, rifapentine, rifaximin), β-lactams(for example, carbacephems (e.g., loracarbef), carbapenems (for example,biapenem, imipenem, meropenem, panipenem), cephalosporins (for example,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin,cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet,cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide,cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome,cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime,cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin,cephaloridine, cephalosporin, cephalothin, cephapirin sodium,cephradine, pivcefalexin), cephamycins (for example, cefbuperazone,cefmetazole, cefininox, cefotetan, cefoxitin), monobactams (for example,aztreonam, carumonam, tigemonam), oxacephems, flomoxef, moxalactam),penicillins (for example, amdinocillin, amdinocillin pivoxil,amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin,azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillinsodium, carbenicillin, carindacillin, clometocillin, cloxacillin,cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacillin,hetacillin, lenampicillin, metampicillin, methicillin sodium,mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamatehydriodide, penicillin G benethamine, penicillin g benzathine,penicillin g benzhydrylamine, penicillin G calcium, penicillin Ghydrabamine, penicillin G potassium, penicillin G procaine, penicillinN, penicillin O, penicillin V, penicillin V benzathine, penicillin Vhydrabamine, penimepicycline, phenethicillin potassium, piperacillin,pivampicillin, propicillin, quinacillin, sulbenicillin, sultamicillin,talampicillin, temocillin, ticarcillin), other (for example, ritipenem),lincosamides (for example, clindamycin, lincomycin), macrolides (forexample, azithromycin, carbomycin, clarithromycin, dirithromycin,erythromycin, erythromycin acistrate, erythromycin estolate,erythromycin glucoheptonate, erythromycin lactobionate, erythromycinpropionate, erythromycin stearate, josamycin, leucomycins, midecamycins,miokamycin, oleandomycin, primycin, rokitamycin, rosaramicin,roxithromycin, spiramycin, troleandomycin), polypeptides (for example,amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin,fusafungine, gramicidin s, gramicidin(s), mikamycin, polymyxin,pristinamycin, ristocetin, teicoplanin, thiostrepton, tuberactinomycin,tyrocidine, tyrothricin, vancomycin, viomycin, virginiamycin, zincbacitracin), tetracyclines (for example, apicycline, chlortetracycline,clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline,meclocycline, methacycline, minocycline, oxytetracycline,penimepicycline, pipacycline, rolitetracycline, sancycline,tetracycline), and others (e.g., cycloserine, mupirocin, tuberin).Agents of use also include synthetic antibacterials, such as2,4-Diaminopyrimidines (for example, brodimoprim, tetroxoprim,trimethoprim), nitrofurans (for example, furaltadone, furazoliumchloride, nifuradene, nifuratel, nifurfoline, nifurpirinol,nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (forexample, cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin,fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin,nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid,pazufloxacin, pefloxacin, pipemidic acid, piromidic acid, rosoxacin,rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin),sulfonamides (for example, acetyl sulfamethoxypyrazine, benzylsulfamide,chloramine-b, chloramine-t, dichloramine t, mafenide,4′-(methylsulfamoyl)sulfanilanilide, noprylsulfamide,phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine,succinylsulfathiazole, sulfabenzamide, sulfacetamide,sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine,sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole,sulfaguanidine, sulfaguanol, sulfalene, sulfaloxic acid, sulfamerazine,sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine,sulfamethoxazole, sulfamethoxypyridazine, sulfametrole,sulfamidocchrysoidine, sulfamoxole, sulfanilamide, sulfanilylurea,n-sulfanilyl-3,4-xylamide, sulfanitran, sulfaperine, sulfaphenazole,sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole,sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide,sulfisomidine, sulfisoxazole) sulfones (for example, acedapsone,acediasulfone, acetosulfone sodium, dapsone, diathymosulfone,glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid,p-sulfanilylbenzylamine, sulfoxone sodium, thiazolsulfone), and others(for example, clofoctol, hexedine, methenamine, methenamineanhydromethylene-citrate, methenamine hippurate, methenamine mandelate,methenamine sulfosalicylate, nitroxoline, taurolidine, xibornol).

Additional agents of use include antifungal antibiotics such as polyenes(for example, amphotericin B, candicidin, dennostatin, filipin,fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin,nystatin, pecilocin, perimycin), others (for example, azaserine,griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin,siccanin, tubercidin, viridin) allylamines (for example, butenafine,naftifine, terbinafine), imidazoles (for example, bifonazole,butoconazole, chlordantoin, chlormiidazole, cloconazole, clotrimazole,econazole, enilconazole, fenticonazole, flutrimazole, isoconazole,ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazolenitrate, sertaconazole, sulconazole, tioconazole), thiocarbamates (forexample, tolciclate, tolindate, tolnaftate), triazoles (for example,fluconazole, itraconazole, saperconazole, terconazole) others (forexample, acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide,buclosamide, calcium propionate, chlorphenesin, ciclopirox, cloxyquin,coparaffinate, diamthazole dihydrochloride, exalamide, flucytosine,halethazole, hexetidine, loflucarban, nifuratel, potassium iodide,propionic acid, pyrithione, salicylanilide, sodium propionate,sulbentine, tenonitrozole, triacetin, ujothion, undecylenic acid, zincpropionate). Antineoplastic agents can also be of use including (1)antibiotics and analogs (for example, aclacinomycins, actinomycin,anthramycin, azaserine, bleomycins, cactinomycin, carubicin,carzinophilin, chromomycins, dactinomycin, daunorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin,menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines,peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin,streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), (2)antimetabolites such as folic acid analogs (for example, denopterin,edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®,trimetrexate), (3) purine analogs (for example, cladribine, fludarabine,6-mercaptopurine, thiamiprine, thioguanine), (4) pyrimidine analogs (forexample, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil,gemcitabine, tagafur).

Steroidal anti-inflammatory agents can also be included such as21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, cyclosporine, deflazacort, desonide, desoximetasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin butyl, fluocortolone,fluorometholone, fluperolone acetate, fluprednidene acetate,fluprednisolone, flurandrenolide, fluticasone propionate, formocortal,halcinonide, halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, and triamcinolonehexacetonide. In addition, non-steroidal anti-inflammatory agents can beused. These include aminoarylcarboxylic acid derivatives (for example,enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamicacid, mefenamic acid, niflumic acid, talniflumate, terofenamate,tolfenamic acid), arylacetic acid derivatives (for example, aceclofenac,acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac,bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac,fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin,isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac,oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin,tropesin, zomepirac), arylbutyric acid derivatives (for example,bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (forexample, clidanac, ketorolac, tinoridine), arylpropionic acidderivatives (for example, alminoprofen, benoxaprofen, bermoprofen,bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen,ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen,oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid,suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (forexample, difenamizole, epirizole), pyrazolones (for example, apazone,benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone,phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone,thiazolinobutazone), salicylic acid derivatives (for example,acetaminosalol, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide o-acetic acid, salicylsulfuric acid, salsalate,sulfasalazine), thiazinecarboxamides (for example, ampiroxicam,droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam),.epsilon.-acetamidocaproic acid, s-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,.alpha.-bisabolol, bucolome, difenpiramide, ditazol, emorfazone,fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol, paranyline,perisoxal, proquazone, superoxide dismutase, tenidap, and zileuton.

Additional Treatment Methods

Methods of treating a subject with ocular inflammation are disclosedherein. The methods include selecting a subject with ocularinflammation, and administering to the subject a therapeuticallyeffective amount of a SLURP1 polypeptide or a nucleic acid encoding theSLURP1 polypeptide. Any of the compositions disclosed herein can be usedin these methods.

In some embodiments, methods of treating ocular inflammation areprovided. In some embodiments, the corneal inflammation treated by themethods disclosed herein are related to ocular disease or an ophthalmicdisorder, such as uveitis, scleritis, episcleritis, keratitis, ocular orophthalmic surgery (e.g., cornea surgery), endophthalmitis, iritis,atrophic macular degeneration, retinitis pigmentosa, iatrogenicretinopathy, retinal tears and holes, cystoid macular edema, diabeticmacular edema, diabetic retinopathy, sickle cell retinopathy, retinalvein and artery occlusion, optic neuropathy, exudative maculardegeneration, neovascular glaucoma, corneal neovascularization,cyclitis, sickle cell retinopathy, pterygium, and contact lenswear-induced conditions, such as a peripheral ulcer. The ocularinflammation can be inflammation of an external surface of the eye, suchas, but not limited to, the cornea, conjunctiva or eye lid.

In some examples, the ocular inflammation results from laser eyetherapy. In other examples, the ocular inflammation results from trauma.In further examples, the ocular inflammation results from exposure toultraviolet light. In yet other examples, the ocular inflammationresults from exposure to chemical stimuli or a toxin. In additionalexamples, the ocular inflammation results from a condition selected fromthe group consisting of Mal de Meleda, allergic conjunctivitis, Reiter'sdisease, scleritis, iridocyclitis, uveitis, Vogt-Koyanagi syndrome,photophthalmia, nongranulomatous inflammation of the uveal tract,granulomatous inflammation of the uveal tract, necrosis of neoplasms,foreign particles lodged in the eye, retinal light toxicity and retinaledema from light exposure. In yet other examples, the ocularinflammation is the result of an infection, such as a viral, bacterialor fungal infection.

The ocular inflammation can be keratitis. In other embodiments, themethods can be used to treat keratitis related to microbial infection.In one specific non-liming example, methods are provided for treatingkeratitis caused by various microbial infections such as gram-negativebacteria (P. aeruginosa and S. marcesans), gram positive bacteria (forexample, S. aureus, S. epidermidis and Corynebacterium species (P.acnes)). In some embodiments, the keratitis is caused by gram positivecocci, gram negative bacilli, gram negative coccobacilli, gram positivebacilli, spirochetes, mycobacteria, or actinomycetes. Therefore, methodsare provided for treating inflammation of the cornea associated withbacterial keratitis. In other embodiments, methods are provided fortreating keratitis caused by a virus, such as an adenovirus, a herpesvirus, a poxvirus, or a rubeola virus. Methods are also provided totreat inflammation of the cornea as a result of a fungal infection. Morespecifically, the methods disclosed herein can be used to treat cornealinflammation caused by an infection with, for example, Fusarium,Penicillium, Aspergillus, Cephalosporium (Acremonium), Trichophyton,Microsporum, Epidermophyton, Scopulariopsis, and Candida. Methods arealso provided for the treatment of inflammation of the cornea frominfection with parasites, such as Onchocerca volvulus, Acanthamoebacasterllani, Acantomoeba polyphagia, Leishmania brasillensis, Onchocercavolvulus, or Trypanosoma sp.

The methods described herein also can be used to treat sterile keratitisin which no living organisms are recovered from either a contact lens orthe corneal surface. In some embodiments, the corneal inflammation isfrom ultraviolet light or other environmental exposure, trauma, or dryeye. The corneal inflammation can be the result of an autoimmunedisease, such as Fuch's superficial marginal keratitis, rheumatoidarthritis, systemic lupus erythematosus, clearoderma, Wegner'sgranulomatosis, polyarteritis nodosa, relapsing polychondritis, Behcetsyndrome, or inflammatory bowel disease.

In additional embodiments, keratitis can be treated in a subject,wherein the inflammation is associated with contact lens wear. Thesesyndromes can include, but are not limited to Contact Lens AssociatedCorneal Infiltrates (CLACI), Contact Lens Associated Red Eye (CLARE),Contact Lens Peripheral Ulcer (CPLU). Sterile and infectious infiltratescan usually, but not always, be distinguished by slit lamp examinationby those having ordinary skill in the art.

Methods are also provided for treating conjunctivitis. Theconjunctivitis can be conjunctivitis from an infectious agent, such as avirus. Viral conjunctivitis can be caused by an adenovirus, a herpessimplex virus, an enterovirus or a coxsackievirus, amongst others.Bacterial conjunctivitis can be caused by S. aureus, S. pneumoniae or H.influenzae, amongst others. The conjunctivitis can be caused by N.gonorrhoeae or N. meningitidis.

In specific non-limiting examples, the conjunctivitis is chronicbacterial conjunctivitis, such as conjunctivitis caused by S. aureus, M.lacunata or other gram-negative enteric bacteria. The conjunctivitis canbe associated with blepharitis.

The conjunctivitis can be chemically induced, such as from theintroduction of an irritant, for example from the introduction of anacid or alkali substance into the eye. The conjunctivitis can beallergic conjunctivitis. Methods are also provided for treatingblepharoconjunctivitis and keratoconjunctivitis. In specificnon-limiting examples the keratoconjunctivitis is keratoconjunctivitissicca, vernal keratoconjunctivitis, atopic keratoconjunctivitis,infectious bovine keratoconjunctivitis, superior limbickeratoconjunctivitis, or keratoconjunctivitis photoelectrica.

The ocular inflammation can be blepharitis. In specific non-limitingexamples, the blepharitis is seborrhoeic, staphylococcal, mixed,posterior or meibomitis, or parasitic. The blepharitis can be posteriorblepharitis or anterior blepharitis.

In other embodiments, the ocular inflammation can be uveitis. Thus, amethod is disclosed herein for the treatment of uveitis in a subject byadministering a therapeutically effective amount of SLURP1 or a nucleicacid encoding SLURP1, thereby treating the subject. Any form of uveitiscan be treated using SLURP1 or a nucleic acid encoding Slurp1. Forexample, iritis, cyclitis, panuveits, iridocyclitis, posterior uveitis,anterior uveitis and diffuse uveitis can be treated using the methodsdisclosed herein. Anterior and/or posterior uveitis can be treated usingSlurp1 or a nucleic acid encoding Slurp1. Both acute onset uveitis andchronic uveitis also can be treated.

In one embodiment, a method is provided for treating anterior uveitis ina subject. Subjects can be treated that are affected with idiopathiciridocyclitis, HLA-B27 positive iridocyclitis, uveitis associated withjuvenile rheumatoid arthritis, Fuch's heterochromatice iridocyclitis,herpes simplex keatovueitis, ankylosing spondylitis, intraocular lensrelated uveitis, Reiter's syndrome, Herpes zoster keratouveitis, uveitisassociated with syphilis, traumatic iridocyclitis, uveitis associatedwith inflammatory bowel disease, tuberculosis iridocyclitis.

In another embodiment, a method is provided for treating posterioruveitis in a subject. Thus subjects can be treated that are affectedwith toxoplasma retinochroiditis, retinal vasculitis, idiopathicposterior uveitis, ocular histoplasmosis, toxocariasis, cytomegalovirusretinitis, idiopathic retinitis, serpinous choroidopathy, acutemultifocal placoid, pigment eptitheliopathy, acute retinal necrosis,bird shot choroidopathy, uveitis associated with a leukemia or alymphoma, reticulum cell sarcoma, ocular candidiasis, tuberculousuveitis, lupus retinitis.

In a further embodiment, a method is provided for treating diffuseuveitis. Thus, subjects can be treated that are affected withsarcoidosis, syphilis, Vogt-Koyanagi-Harada syndrome, or Bechet'sdisease.

In one embodiment, a sign or a symptom of the uveitis is decreased oralleviated. Ocular signs include ciliary injection, aqueous flare, theaccumulation of cells visible on ophthalmic examination, such as aqueouscells, retrolental cells, and vitreous cells, keratic precipitates, andhypema. Symptoms include pain (such as ciliary spasm), redness,photophobia, increased lacrimation, and decreased vision. One of skillin the art can readily diagnose uveitis. In one embodiment,biomicroscopy (for example, a “slit lamp”) is used to diagnose uveitis,to evaluate the clinical course of the disease or to verify that atreatment protocol has been successful.

In additional embodiments, the subject can be administered an additionalpharmaceutical agent, such as a steroidal or non-steroidalanti-inflammatory agent, anti-bacterial agents, anti-fungal agents, oranti-neoplastic agent. The phrase “combinatorial therapy” or“combination therapy” embraces the administration of a SLURP1polypeptide and/or a nucleic acid encoding the SLURP1 polypeptide, andone or more therapeutic agents as part of a specific treatment regimenintended to provide beneficial effect from the co-action of thesetherapeutic agents. Administration of these therapeutic agents incombination typically is carried out over a defined period (usuallyminutes, hours, days or weeks depending upon the combination selected).“Combinatorial therapy” or “combination therapy” is intended to embraceadministration of these therapeutic agents in a sequential manner, thatis, wherein each therapeutic agent is administered at a different time,as well as administration of these therapeutic agents, or at least twoof the therapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample by administering to the subject an individual dose having afixed ratio of each therapeutic agent or in multiple, individual dosesfor each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, topicaladministration, oral routes, intravenous routes, intramuscular routes,and direct absorption through mucous membrane tissue. The therapeuticagents can be administered by the same route or by different routes. Anyof the compositions disclosed above can be used in the presently claimedmethods.

In some embodiments, combinational therapy can include theadministration of a SLURP1 polypeptide, and/or a nucleic acid encodingthe SLURP1 polypeptide, with at least one antibacterial, antiviral orantifungal agent, such as to treat ocular inflammation, such as cornealinflammation. In one specific example, the combinational therapyincludes a SLURP1 polypeptide, and/or a nucleic acid encoding the SLURP1polypeptide, and at least one ophthalmic antibiotic or ophthalmicantiviral. Ophthalmic antibiotics include, for example, chloramphenicolsodium succinate ophthalmic (chloramphenical); CORTISPORIN® (neomycinand polymyxin β sulfates and hydrocortisone acetate cream); ILOTYCIN®(erythromycin ophthalmic ointment); NEODECADRON® (neomycinsulfate-dexamethasone sodium phosphate); POLYTRIM® (trimethoprim andpolythyxin.beta. sulfate ophthalmic solution); TERRA-CORTRIL®(oxytetracycline HCL and hydrocortisone acetate); TERRAMYCIN®(oxytetracycline); and TOBRADEX® (tobramycin and dexamethosoneophthalmic suspension and ointment). Ophthalmic antivirals include, forexample, VIRA-A® ophthalmic ointment, (vidarabine). Ophthalmicquinalones include, for example, CHIBROXIN® (norfloxacin ophthalmicsolution); CILOXAN® ophthalmic solution, (Ciprofloxacin HCL); andOCUFLOX® ophthalmic solution (ofloxacin). Ophthalmic sulfonamidesinclude, for example, BLEPHAMIDE® ophthalmic ointment (sulfacetamidesodium and prednisolone acetate); and BLEPHAMIDE® ophthalmic suspension(sulfacetamide sodium and prednisolone acetate). Antifungals include,for example, natamycin and amphotericin-B.

Methods are provided for treating an inflammatory response in asubject's eye, such as in the cornea, conjunctiva, eye lid, or uvealtract. The method includes administering to the subject atherapeutically effective amount of a SLURP1 polypeptide, and/or anucleic acid encoding the SLURP1 polypeptide. In one aspect of thepresent invention, the treatment can result in the inhibition ofcellular infiltrate into the subject's cornea. More particularly, thetreatment of the inflammatory response can include the inhibition ofneutrophil infiltration. In some embodiments, neutrophil infiltration isreduced by at least about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%.

For any of the methods disclosed herein, the SLURP1 polypeptide, orpolynucleotide encoding the SLURP1 polypeptide, can be administeredsystemically or locally. In some embodiments, the SLURP1 polypeptide, orpolynucleotide encoding the SLURP1 polypeptide, is administered locallyto the eye. The administration can be topical, such as in an ophthalmicsolution or ointment, or in a contact lens placed in the eye. The SLURP1polypeptide, or polynucleotide encoding the SLURP1 polypeptide, can beincluded in an implant that is implanted in the eye. However,administration can also be systemic.

The SLURP1 polypeptide, or a nucleic acid encoding the SLURP1polypeptide, is delivered for a sufficient time period to achieve thedesired effect. Thus, in one embodiment, the SLURP1 polypeptide, or anucleic acid encoding the SLURP1 polypeptide, is delivered for at leastabout 2 days, such as for about five days, seven days, ten days, 14 or21 days. In several embodiments, the SLURP1 polypeptide, or a nucleicacid encoding the SLURP1 polypeptide, is delivered for at least aboutone week, at least about two weeks, at least about three weeks, at leastabout four weeks, at least about five weeks, at least about six weeks,at least about seven weeks, at least about eight weeks, at least aboutnine weeks, at least about 10 weeks, and at least about 12 weeks. Theduration of use of a SLURP1 polypeptide, or a nucleic acid encoding theSLURP1 polypeptide, can be the medical history of the subject and othercontributing factors (such as use of other agents, etc.). If extendedperiods of administration are required, the SLURP1 polypeptide, or anucleic acid encoding the SLURP1 polypeptide, can be administered for upto six months, or one year, or longer. In one embodiment, extendedperiods of delivery are achieved with the use of an implant or a contactlens (see above). Thus, administration can be continuous or repeated.

Any of the administration methods and/or compositions disclosed abovecan be utilized. More than one method of administration also can also beutilized, such as a combination of instillation methods. For example,implants can be sequentially implanted into the vitreous in order tomaintain concentrations for even long periods. In one embodiment, morethan one implant can be sequentially implanted into the eye in order tomaintain therapeutic drug concentrations for longer periods. Implants orcontact lens can be combined with ophthalmic solutions or ointments.Topical administration can also be combined with systemicadministration.

In one embodiment, a sign or a symptom of the inflammation, such ascorneal inflammation is decreased or alleviated. Administration can besystemic or local. One polypeptide or polynucleotides, or multiplepolypeptides and polynucleotides can be utilized.

EXAMPLES

The secreted Ly-6/urokinase plasminogen activator receptor-relatedprotein-1 (SLURP1) belongs to the Ly6/uPAR superfamily of proteins thatparticipate in signal transduction, immune activation and cell adhesionand are characterized by the presence of three-finger structuregenerated by five disulfide bridges (Grando et al., J Pharmacol Sci2008; 106:174-179, Mazar et al., Clin Cancer Res 2008; 14:5649-5655,Adermann et al., Protein Sci 1999; 8:810-819)

SLURP1 is expressed in a variety of cells including immune cells(Moriwaki et al., Life Sci 2007; 80:2365-2368, bronchial epithelialcells, (Horiguchi et al., J Neurosci Res 2009; 87:2740-2747) primarysensory neurons (Moriwaki et al., Neurosci Res 2009; 64:403-412), theskin, exocervix, gums, stomach, trachea and esophagus (Mastrangeli etal., Eur J Dermatol 2003; 13:560-570), oral keratinocytes (Arredondo etal., Life Sci 2007; 80:2243-2247) and the cornea (Norman et al., InvestOphthalmol Vis Sci 2004; 45:429-440). In the skin, Slurp1 is abundantlyexpressed in the keratinocytes underlying the stratum corneum. Slurp1 isone of the most abundant transcripts in the neonatal and the adult mousecorneas (Norman et al., Invest Ophthalmol Vis Sci 2004; 45:429-440).Human SLURP1 mRNA expression is regulated by retinoic acid, epidermalgrowth factor and interferon-γ (Mastrangeli et al., Eur J Dermatol 2003;13:560-570).

SLURP1 is a secreted protein, and is detected in many bodily fluidsincluding the plasma, saliva, sweat, tears and urine, and is considereda late marker of epidermal differentiation (Favre et al., J InvestDermatol 2007; 127:301-308). Slurp1 is thought to fine-tune thephysiologic regulation of keratinocyte functions through the cholinergicpathways, as it is structurally similar to the snake and frog cytotoxina-bungarotoxin, and acts as a ligand for the α7 subunit of the nicotinicacetylcholine receptors (a7nAchRs) (Mastrangeli et al., Eur J Dermatol2003; 13:560-570, Arredondo et al., J Invest Dermatol 2005;125:1236-1241). Slurp1 is involved in signal transduction, activation ofthe immune response, and cell adhesion, preventing tobacconitrosamine-induced malignant transformation of oral cells. (Grando etal., J Pharmacol Sci 2008; 106:174-179, Moriwaki et al., Life Sci 2007;80:2365-2368, Arredondo et al., Life Sci 2007; 80:2243-2247, Arredondoet al., Biochem Pharmacol 2007; 74:1315-1319, Chimienti et al., Hum MolGenet 2003; 12:3017-3024). The secreted Ly6/uPAR-related protein-1(SLURP1) is associated with the hyperkeratotic disorder Mal-de-Meleda.

The expression and function of Slurp1 in the corneas was evaluated andis disclosed herein. Gene expression was quantified by qPCR, immunoblotsand immunofluorescent staining. Effect of Klf4 on Slurp1 promoter wasevaluated by chromatin immunoprecipitation (ChIP) and transienttransfections. Adenoviral vectors were used to express Slurp1 incorneas. Leukocytic infiltration in bacterial lipopolysaccharides (LPS),Herpes Simplex Virus Type-1 (HSV-1) or adenovirus (serotype-5) treatedmouse corneas was characterized by flow cytometry.

As shown below, corneal expression of Slurp1 increased sharply uponmouse eyelid opening, concurrent with the elevated expression of Klf4.Slurp1 was significantly decreased in Klf4-conditional null (Klf4CN)corneas which displayed elevated expression of cytokines and cytokinereceptors, and neutrophil influx consistent with a pro-inflammatoryenvironment. In additional models of corneal inflammation, Slurp1expression was abrogated within 24 hours (h) of bacterial LPS injection,HSV-1 or adenoviral infection, accompanied by a predominantlyneutrophilic infiltrate. Neutrophilic infiltration was enhanced in HSV-1infected Klf4CN corneas lacking Slurp1. Slurp1 promoter activity wasstimulated by KLF4, suppressed by IL-4, IL-13 and TNFα, and unperturbedby interferon-γ (IFN-γ). Slurp1 downregulation and neutrophil influxwere comparable in HSV-1 infected wild type (WT) and IFNγ −/− mousecorneas. Mouse corneas infected with Slurp1-expressing adenoviralvectors displayed reduced signs of inflammation and restrictedneutrophilic infiltration compared with those infected with controlvectors.

The results demonstrated that Klf4 regulates the expression of Slurp1, akey immunomodulatory peptide that is abundantly expressed in healthycorneas and is downregulated in pro-inflammatory conditions.Furthermore, SLURP1 can be used therapeutically as an immunomodulatorymolecule to treat ocular inflammation, such as, but not limited to,keratitis.

Example 1 Materials and Methods

Mice.

Klf4CN mice were generated on a mixed background by matingKlf4^(loxP/loxP), Le-Cre/− mice with Klf4^(loxP/loxP) mice to obtainroughly equal proportion of Klf4^(loxP/loxP), Le-Cre/− (Klf4CN) andKlf4^(loxP/loxP) (WT control siblings) offspring as described earlier(Swamynathan et al., Mol Cell Biol 2007; 27:182-194). Wild type andIFN-γ knockout (GKO) BALB/c mice were purchased.

LPS Injection, HSV-1 or Adenoviral Infection of Mouse Corneas.

Mice were anesthetized by intraperitoneal injection of 2.0 mg ofketamine hydrochloride and 0.04 mg of xylazine (Phoenix Scientific, St.Joseph, Mo.) in 0.2 ml of Hanks balanced salt solution (Cambrex, CharlesCity, Iowa). The corneas of anesthetized mice were abraded 10 times in acrisscross fashion followed by topical application of 3 μl of RPMI 1640(Cambrex, Charles City, Iowa) alone (mock infected) or RPMI 1640containing 1×10⁵ plaque forming units (PFU) of wild-type HSV-1 RE (HSV-1infected) (Sheridan et. al., J Virol 2009; 83:2237-2245) Intrastromalinjections of ultrapure bacterial LPS (Sigma-Aldrich Co., St. Louis,Mo.; 20 mg in 2 ml sterile water/cornea) were performed using finetipped Hamilton syringes in tunnels generated by 32 gauge syringeneedles.

Adenoviral vectors expressing Slurp1 were constructed in ADENOX®expression system (Serotype-5; Clontech Laboratories, Mountain View,Calif.) and amplified in HEK293 cells. 2×10⁶ PFU of Adv5-Tet-Off alone(Control) or 10⁶ PFU each of Adv5-Tet-Off and Adv5-Slurp1 were used perabraded cornea, in mice anesthetized as above. Slit-lamp biomicroscopeimages were collected from anesthetized mice using SL-130 slit lamp(Carl Zeiss Meditec, Dublin, Calif.) equipped with a digital camera.Corneas were harvested at 4 days post-infection (DPI), and immersed in1× phosphate buffered saline (PBS)-EDTA for 15 minutes at 37° C. toremove overlying epithelium, for isolating total RNA for quantitativepolymerase chain reaction (QPCR). Stromal leukocytes were isolated,labeled and characterized by flow cytometry as described below.

Isolation of Total RNA and qPCR.

Corneas were excised from normal (non-infected) mice or at 6 hours (h),12 h, 24 h, or 48 h after mock infection or HSV-1 corneal infection andsoaked in RNA-Later to preserve the RNA integrity. Total RNA wasisolated using the RNEASY® Mini kit (Qiagen, Valencia, Calif.). Unlessotherwise mentioned, Applied Biosystems (Foster City, Calif.) was thesource for the reagents, equipment and software for TAQMAN® geneexpression quantitative real time PCR assays (qPCR). QPCR assays withpre-standardized gene-specific probes for different transcripts wereperformed in ABI STEPONE PLUS® thermal cycler using 18S rRNA orlaminin-B1 as endogenous controls (to avoid skewing of the results dueto the 18S rRNA from infiltrating immune cells in the inflamed corneas).Expression levels of different cytokines were quantified using the mousecytokines PCR array following the protocol suggested by the manufacturer(Super-Array Biosystems, Frederick, Md.).

Effect of Cytokines on KLF4 and SLURP1 Expression.

cDNA was synthesized using 1 mg total RNA isolated from HCLE cells(Gipson et al., Invest Ophthalmol Vis Sci 2003; 44:2496-2506) treatedwith IL-2 (R and D Systems, Minneapolis, Minn.; 1 ng/ml), IL-4(PeproTech, Rocky Hill, N.J.; 0.5 ng/ml), IL-13 (PeproTech, Rocky Hill,Rocky Hill, N.J.; 5 ng/ml), IFN-γ (Chemicon, Billerica, Mass.; 1 ng/ml),or TNFa (Fisher Scientific, Pittsburgh, Pa.; 0.2 ng/ml) for 2 days. QPCRwas performed with KLF4- and SLURP1-specific probes and TAQMAN® reagents(Applied Biosystems, Carlsbad, Calif.) using 18S rRNA as endogenouscontrol.

Immunoblots.

Equal amounts of total protein extracted by homogenizing dissectedcorneas in urea lysis buffer (8.0 M urea, 0.08% Triton X-100, 0.2%sodium dodecyl sulfate, 3% β-mercaptoethanol, and proteinase inhibitors)and quantified by the bicinchoninic acid method (Pierce, Rockford, Ill.)were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels,transferred to polyvinylidene difluoride membranes and subjected toimmunoblot analysis. Rabbit anti-mouse Slurp1 Moriwaki Y, Watanabe Y,Shinagawa T, et al. Primary sensory neuronal expression of SLURP-1, anendogenous nicotinic acetylcholine receptor ligand. Neurosci Res 2009;64:403-412 (Moriwaki et al., Neurosci Res 2009; 64:403-412) (1:1000dilution) and goat anti-actin (Santa Cruz Biotechnology, Santa Cruz,Calif.) (1:1000 dilution) antibody were used as primary antibodies inPBS-Tris (PBST). Horseradish peroxidase-coupled goat anti-rabbit IgG(Invitrogen, Carlsbad, Calif.) (1:2000 dilution) or rabbit anti-goat IgG(Kirkegarad and Perry, Gaithersburg, Md.) (1:5000 dilution) was used assecondary antibody. Immunoreactive bands were detected bychemiluminescence using Super Signal West Pico solutions (Pierce,Rockford, Ill.).

Immunofluorescent Staining of Corneal Cryosections.

8-10 μm-thick cryosections from OCT-embedded mouse eyes or human centralcorneas were fixed in fresh 4% paraformaldehyde in PBS for 30 min,blocked for 1 h at room temperature with 10% goat serum in PBST, washedtwice with PBST for 5 min each, incubated overnight at 4° C. with a1:500 dilution of rabbit anti-mouse Slurp1 primary antibody (Moriwaki etal., Life Sci 2007; 80:2365-2368) (Moriwaki et al., Neurosci Res 2009;64:403-412) or 1:50 dilution of goat anti-human SLURP1 antibody (SantaCruz Biotechnology, Santa Cruz, Calif.), washed thrice with PBST for 10min each, incubated with secondary antibody (AlexaFluor 546-coupled goatanti-rabbit IgG or Alexafluor 488-coupled donkey anti-goat IgG;Molecular Probes, Carlsbad, Calif.) at a 1:500 dilution for 1 h at roomtemperature, rinsed with PBS, incubated with DAPI for 10 minutes, washedthrice with PBST for 5 min each, mounted with Aqua Polymount(Polysciences, Inc), observed and images collected with Olympus IX81microscope and Fluoview 1000 confocal system. All images presentedwithin each composite figure were acquired under identical settings andprocessed in a similar manner using Adobe Photoshop and Illustrator(Adobe, Mountain View, Calif.).

Immunofluorescent Staining of Corneal Whole Mounts.

Corneas were dissected, flattened by three radial incisions, washedthree times for 15 minutes each in PBS with 4% fetal bovine serum (FBS)and blocked in Fc block (BD Pharmingen, San Jose, Calif.) for 20 minutesprior to incubation with fluorescein isothiocyanate (FITC)-conjugatedanti-CD45 antibody (BD Pharmingen, San Jose, Calif.) overnight at 4° C.Corneas were then washed three times each for 30 minutes in PBS/4% FBS,fixed in 1% Paraformaldehyde for 2 hours at 4° C., rinsed three timesagain for 30 minutes each in PBS/4% FBS and mounted in Aqua poly/Mount(Polysciences Inc, Warrington, Pa.). Images were acquired on an OlympusFluoview 1000 confocal system with an Olympus IX81 microscope. Stackswere imaged at 20× (numerical aperture (NA) 0.85) and 60× (NA 1.42) andmaximum intensity projections were imaged through the stromal portion ofthe stack.

Flow Cytometry.

Corneas were excised 48 h after mock or HSV-1 corneal infection, andimmersed in 1× PBS-EDTA for 15 minutes at 37° C. to remove overlyingepithelium. The corneal stroma was digested in collagenase type 1 (840U/cornea, Sigma-Aldrich Co. St. Louis, Mo.) for 1 hour at 37° C. Singlecell suspensions were generated by trituration and filtered through a40-μm cell strainer cap (BD Labware, Bedford, Mass.). Suspensions wereincubated with anti-mouse CD16/CD32 (Fcγ receptor; clone 2.4G2; BDPharmingen, San Diego, Calif.), and then stained withfluorochrome-conjugated antibodies to various surface markers for 30minutes at 4° C. The following antibodies and their correspondingisotypes were used for phenotypic analysis: PerCP-conjugated anti-CD45(clone 30-F11; BD Pharmingen, San Diego, Calif.), APC-conjugatedanti-Gr-1(clone RB6-8C5; Caltag Laboratories, Burlingame, Calif.), andeFluor450-conjugated anti-CD11b (clone M1/70; eBioscience, San Diego,Calif.). After staining, cells were fixed with 1% paraformaldehyde andmixed with COUNTBRIGHT® absolute counting beads (10,000 beads/sample;Invitrogen, Carlsbad, Calif.). Data were collected on a FACSARIA®cytometer and analyzed by FACSDIVA® Software (BD Biosciences, San Jose,Calif.). A gate was established to stop acquiring events aftercollecting 80% of the beads. The absolute number of each cell type percornea was determined by calculating the number of events shown in thegate, multiplied by a factor of 1.25.

Chromatin Immunoprecipitation (ChIP).

ChIP was based on the EZ-CHIP® protocol (Upstate, Inc, Charlottesville,Va.). DNA-bound proteins were cross linked with 1% paraformaldehyde, thechromatin purified, sonicated and immunoprecipitated with pre-immuneserum or anti-KLF4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.) and protein G-sepharose. Following reversal of cross linking byheating overnight at 65° C. in the presence of NaCl and purification ofeluted DNA, Slurp1 promoter fragments were detected by PCR with -396F(5′ TTTATCAGGC AGGCAGATAT AAAGC 3′, SEQ ID NO: 5) and +30R (5′ATTCTTCAGT GCTCAGGAGC T 3′, SEQ ID NO: 6) primers.

Reporter Vectors, Cell Culture, and Promoter Activities.

Reporter vectors were generated in pGL3Basic (Promega, Madison, Wis.) bycloning the SLURP1 promoter fragments amplified using the followingprimers: −500F: 5′-ACA TCA GGT ACT CCC TCC T-3′, -150F: 5′-GGC CCC ACCCTG GGA TGG TAG GTG A-3′, and +30R: 5′-TCT TCA GTG CTC AGG AGC TAGGA-3′. Transient expression of Klf4 was achieved using CMV promoter inpCI-Klf4. Human keratinocyte NCTC cells and SV40-transformed cornealepithelial (HCE) cells (Araki-Sasaki et al., Invest Ophthalmol Vis Sci1995; 36:614-621) were grown in six-well plates as described earlier(Swamynathan, et al., Invest Ophthalmol Vis Sci 2011; 52:1762-1769) andtransfected with 0.5 μg reporter vector pSlurp1-Luc, 10 ng pRL-SV40(Promega, Madison Wis., for normalization of transfection efficiency)and 0.5 μg pCI or pCI-Klf4, using 3 μl Fugene 6 (Roche MolecularBiochemicals, Indianapolis, Ind.). Transient knockdown of KLF4expression in human corneal limbal epithelial (HCLE) (Gipson et al.,Invest Ophthalmol Vis Sci 2003; 44:2496-2506) cells was achieved usingplasmids expressing Anti-KLF4 siRNA described earlier (SuperArrayBiosciences, Frederick, Md.) (Swamynathan et al., Invest Ophthalmol VisSci 2011; 52:1762-1769) HCLE cells in six-well plates wereco-transfected with 1.0 μg control or anti-KLF4 siRNA plasmid, 0.5 μgreporter vector (−500/+27 bp Slurp1-Luc) and 15 ng pRL-SV40 (Promega,Madison Wis., for normalization of transfection efficiency) using 4.5 μlLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). After two days oftransfection, cells were lysed with 500 μl of passive lysis buffer and50 μg protein in the supernatant was analyzed using a dual-luciferaseassay kit (Promega, Madison Wis.) and a Synergy-II microplateluminometer (Biotek Instruments, Winooski, Vt.) as earlier. (Swamynathanet al., Invest Ophthalmol Vis Sci 2011; 52:1762-1769) Results from threeindependent experiments, normalized for transfection efficiency usingthe SV40 promoter-driven Renilla luciferase activity, were used toobtain mean promoter activities and standard deviation. Fold-activationwas determined by dividing mean promoter activity in the presence ofpCI-Klf4 by the promoter activity in the absence of pCI-Klf4.

Example 2 Corneal Expression of Slurp1

QPCRs revealed, and immunofluorescent staining confirmed that Slurp1expression is increased by more than 15-fold between postnatal day 11(PN11) and PN21 (FIGS. 1A and B), suggesting a critical role for Slurp1in post-eyelid opening stages when the cornea is first exposed to theenvironment. Though Slurp1 was detected in all layers of the cornea, itsexpression was much higher in the epithelium (FIG. 1B), consistent withthe previous in situ hybridization data (Norman et. al., InvestOphthalmol Vis Sci 2004; 45:429-440). The spatial distribution of SLURP1in human corneas was comparable to that in the mouse corneas, albeitwith a little higher expression in the stroma, as demonstrated byimmunofluorescent staining of human corneal sections from a healthy 52year-old male organ donor (FIG. 1C). Together, these results reveal thatSlurp1 is expressed at high levels in mature mouse and human corneas,with comparable tissue distribution (FIG. 1).

Example 3 Slurp1 and the Ocular Surface

The effect of Slurp1 on the functions of membrane-tethered uPAR, such asby acting as a soluble scavenger of its ligand urokinase-typeplasminogen activator (uPA) was evaluated. It was also evaluated whetherhuman SLURP1 expression is dependent on ocular surfaces health.

For these studies, recombinant 6× His-Slurp1 and MBP-uPA were expressedin E. coli and partially purified using nickel-ion and amylose columns,respectively. The interaction of Slurp1 with uPA was detected usingligand blots, enzyme-linked immunosorbent assays (ELISA), pull-downassays and immunofluorescent staining. Mouse corneal stromal fibroblastM/KT-1 cells were employed to examine the effect of Slurp1 on cellproliferation, migration, and attachment to extracellular matrix (ECM).Expression of Slurp1 in human tears from healthy or inflamed ocularsurface was assessed by immunoblots.

The ligand blots, ELISA, and pull-down assays indicated that Slurp1efficiently interacts with uPA. Immunofluorescent staining demonstratedthat exogenous Slurp1 decreased the amount of cell surface-bound uPApresent in discrete foci in stagnant cells, and in the leading edge ofmigrating cells. M/KT-1 cell proliferation, ECM attachment, andmigration rate were suppressed by Slurp1. SLURP1 was abundant in tearsfrom healthy human ocular surface and was either decreased or absent intears from inflamed ocular surface.

Without being bound by theory, Slurp1 can modulate corneal inflammationby serving as a soluble scavenger of uPA, and regulating the ocularsurface functions of uPAR. Furthermore, the results confirmed thatSLURP1 is active in humans.

Example 4 Klf4 Binds and Upregulates Slurp1 Promoter Activity

The increase in Slurp1 expression during post-eyelid opening stages isconcurrent with an increase in the expression of Klf4 (FIG. 2A) whichplays critical roles in maturation and maintenance of the mouse ocularsurface, (Swamynathan et al., Invest Ophthalmol Vis Sci 2011;52:1762-1769, Swamynathan et al., Invest Ophthalmol Vis Sci 2008;49:3360-3370, Swamynathan et al., Mol Cell Biol 2007; 27:182-194)raising the possibility that Klf4 regulates Slurp1 expression.Microarray data (Swamynathan et al., Invest Ophthalmol Vis Sci 2008;49:3360-3370), and the present quantitative polymerase chain reaction(QPCR), immunoblots and immunofluorescent staining confirmed asignificant decrease in Slurp1 expression in Klf4CN compared with the WTcorneas (FIGS. 2B, C, and D). These results suggest that Klf4 regulatesthe sharp increase in post-eyelid opening expression of Slurp1.

In order to directly test if Klf4 regulates Slurp1 promoter activity,the luciferase reporter vectors driven by mouse Slurp1 promoter(−500/+27 base pair (bp) or −150/+27 bp fragments; FIG. 3A) wasco-transfected with increasing amounts of the empty control vector pCIor the expression vector pCI-Klf4, in human corneal epithelial (HCE) orskin keratinocyte (NCTC) cells. Activities of both −500/+27 bp and−150/+27 bp Slurp1 promoter fragments were increased uponco-transfection with pCI-Klf4 (FIGS. 3B and C), suggesting that theKlf4-responsive elements are located within the −150/+27 bp Slurp1proximal promoter. In addition, specific siRNA-mediated knockdown ofKLF4 resulted in reduced −500/+27 bp Slurp1 promoter activity, relativeto that obtained with co-transfection of control siRNA-expressingplasmids in HCLE cells (FIG. 3D). Chromatin immunoprecipitation (ChIP)assays demonstrated that the −396/+30 bp Slurp1 promoter fragment isbound by KLF4 in HCE cells (FIG. 3E). Finally, examination of the Slurp1promoter sequence revealed the presence of several potentialKLF4-binding sites (GC-rich regions with a core sequence CACCC⁴¹) withinthe −500/+27 bp fragment, many clustered within the −150/+27 bp region(FIG. 3F), consistent with the stimulation of the −150/+27 bp proximalpromoter activity by Klf4. Taken together, these results demonstratethat Klf4 binds and upregulates Slurp1 proximal promoter activity.

Example 5 Inflammatory Environment in Klf4CN Corneas

Deletions or mutations in SLURP1 cause autosomal recessive inflammatorydisorder Mal-de-Meleda (Mastrangeli et al., Eur J Dermatol 2003;13:560-570, Favre et al., J Invest Dermatol 2007; 127:301-308, Arredondoet al., Biochem Pharmacol 2007; 74:1315-1319, Chimienti et al., Hum MolGenet 2003; 12:3017-3024, Fischer et al., Hum Mol Genet 2001;10:875-880, Eckl et al., Hum Genet 2003; 112:50-56, Hu et al., J InvestDermatol 2003; 120:967-969, Marrakchi et al., J Invest Dermatol 2003;120:351-355, Ward et al., J Invest Dermatol 2003; 120:96-98). Slurp1expression is decreased in diverse pro-inflammatory conditions includingsuture- or alkali burn-induced corneal neovascularization (Jia et al.,Mol Vis 2011; 17:2386-2399) (NCBI GEO accession number GSE23347),asthmatic lungs (Narumoto et al., Biochem Biophys Res Commun 2010;398:713-718), Barrett's esophagus, adenocarcinomas, malignant melanomas,cervical cancer, and oral squamous cell carcinomas, (NCBI GEO AccessionNumbers GDS1321, GDS3472, GDS1375 and GDS1584). It was tested if downregulation of Slurp1 is accompanied by pro-inflammatory conditions inKlf4CN corneas. QPCR revealed that the expression of interferon-γ, 19different chemokines, 8 chemokine receptors, 5 interleukins, and 5interleukin receptors is up-regulated by more than 4-fold in the Klf4CNcompared with the WT corneas (Table 1), indicating a pro-inflammatoryenvironment in Klf4CN corneas. Table 1

Real Time Q-RT-PCR Estimation of the Expression Levels of Cytokines,Chemokine Ligands and Chemokine Receptors in the Klf4CN Cornea

A. Cytokines/Chemokine Ligands

Relative Expression in Klf4CN Target Cells Gene cornea (Chemoattractantfor) Il13 19.126 Il4 6.904 Il1f6 5.380 Il3 5.054 Il10 5.342 1fng 5.089Cxcl5 12.707 Neutrophils Cxcl11 9.563 Activated T cells Cxcl15 8.923Neutrophils Ccl12 8.268 Peripheral blood monocytes Ccl19 7.931 Dendriticcells Ccl11 7.822 Eosinophils Cxcl10 7.555 Macrophages, T cells, NKcells, and dendritic cells Ccl1 6.904 Monocytes, NK cells, immature Bcells and dendritic cells Ccl6 6.353 Macrophages, B cells, CD4 T cells,eosinophils Cxcl9 6.309 Activated T cells and NK cells Ccl8 5.969 Mastcells, eosinophils, basophils, monocytes, T cells, and NK cells Ccl205.726 Dendritic cells Ccl5 5.531 T cells, eosinophils, and basophilsCxcl13 5.269 B cells Ccl17 5.232 Activated T cells Ccl22 4.985 Dendriticcells, NK cells, and Th2 cells Ccl7 4.882 Monocytes Cxcl12 4.749Lymphocytes Pf4 4.250 Neutrophils and monocytes

B. Chemokine Receptors

Relative Expression Gene in Klf4CN cornea Ccr7 15.322 Il1r2 14.395 Ccr48.325 Il5ra 8.325 Ccr1 7.001 Ccr6 6.487 Ccr2 5.726 Il10ra 5.342 Ccr35.342 Cxcr5 5.306 Xcr1 5.160 Il8rb 4.221 Il2rg 4.192 Cd40lg 12.884 Itgb211.856

Furthermore, Klf4CN corneas displayed a significantly higher density ofbone marrow-derived CD45⁺ cells in comparison with the WT corneas (FIG.4). While CD45⁺ cells were sparsely and evenly distributed throughoutthe WT corneal stromas, they were present in large numbers in discreteclusters in the Klf4CN corneas (FIG. 4). Thus, Klf4CN corneas lackingSlurp1 exhibit a marked pro-inflammatory environment.

Example 6 Slurp1 Expression is Reduced in Inflamed Corneas

In order to determine if reduced expression of Slurp1 is a common themein inflamed corneas, additional models of corneal inflammation includingHerpes Simplex Virus Type-1 (HSV-1) infection and bacteriallipopolysaccharide (LPS) injection were examined. Slurp1 was sharplydecreased one day after HSV-1 infection or LPS-injection, while Klf4 wasnot affected (FIG. 5). Klf4 was only partially reduced after two days inHSV-1 infected corneas, suggesting that the rapid reduction in Slurp1levels within 24 h of LPS or HSV-1 treatment is not due to inadequateKlf4 levels (FIG. 5) Immunofluorescent staining confirmed the decreasedexpression of Slurp1 in HSV-1 infected mouse corneas at both 1 and 2DPI, and revealed that much of the decrease occurs within the cornealepithelium (FIG. 5B). Along with a decrease in Slurp1 expression, agreater influx of cells stained with DAPI was also observed after HSV-1infection. Taken together, these results demonstrate that the decreasedexpression of Slurp1 is a common theme in inflammation independent ofthe nature of insults, and suggest an immunomodulatory role for Slurp1.

Example 7 Reduced Expression of Slurp1 is Associated with IncreasedNeutrophilic Infiltration

Flow cytometric analysis 2 days after mock infection revealedsignificantly (p=0.02) higher frequency of CD45⁺ cells in the Klf4CNcompared with the WT corneas (FIGS. 6A and B). The Klf4CN corneal CD45⁺cells were predominantly neutrophilic in nature (CD11b⁺ Gr-1⁺) comparedwith the predominantly macrophage phenotype (CD11b⁺ Gr-1⁻) of wild type(WT) corneal CD45⁺ cells (FIGS. 6C and D). At 2 DPI, both WT and Klf4CNcorneas exhibited elevated infiltration of bone marrow-derived CD45⁺cells comprised predominantly of CD11b⁺ Gr-1⁺ neutrophils, the frequencyof which was significantly higher (p<0.0001) in the Klf4CN corneas (FIG.6). Although it did not reach statistical significance, there was atrend towards higher absolute numbers of neutrophils in the infectedKlf4CN corneas (Table 2A).

TABLE 2 Enumeration of CD45+, and Gr-1+ CD11b+ cells Gr-1+ CD11b+Treatment CD45+ Cells ± SEM Cells ± SEM (A) Abraded and HSV-1-infectedWT and Klf4CN corneas WT Abraded  210 ± 65   41 ± 14 WT Infected  1350 ±361  1310 ± 356 Klf4CN  455 ± 160 (p = 0.1938)  403 ± 157 (p = 0.0507)Abraded Klf4CN  8687 ± 3793 (p = 0.0903)  8529 ± 3733 (p = 0.0904)Infected (B) Abraded and HSV-1-infected WT and GKO corneas WT Abraded 1997 ± 394  445 ± 129 WT Infected 12283 ± 1677  9729 ± 2730 GKO Abraded 1994 ± 355 (p = 0.9956)  686 ± 201 (p = 0.3425) GKO Infected 13125 ±2443 (p = 0.7835) 11096 ± 2319 (p = 0.7127) CD45+, and Gr-1+ CD11b+cells were enumerated in abraded and HSV-1-infected (A) WT and Klf4CNcorneas (mixed background), and (B) WT and GKO corneas (BALB/c). Averagenumber of cells derived from three independent replicates is providedwith standard error of mean (SEM). The p values provide a measure ofstatistical significance in comparison with the corresponding WTsamples. The differences in CD45+ and Gr-1+ CD11b+ cell numbers in theWT abraded and infected corneas between the two experiments may be dueto the different strains used, and/or different size of the corneaanalyzed, to account for the smaller Klf4CN corneas.

Thus, HSV-1 infection overcomes the barrier to neutrophilic infiltrationinto WT corneas, but does so more effectively in Klf4CN corneas lackingSlurp1 from the time of infection, consistent with the immunomodulatoryrole of Slurp1.

Example 8 SLURP1 Expression is Inhibited by Pro-Inflammatory CytokinesIL-4, IL-13 and TNF-α, but not Interferon-γ

Interferon-γ (IFN-γ) and interleukin 13 (IL-13) suppress Slurp1(Mastrangeli et al., Eur J Dermatol 2003; 13:560-570, Narumoto et al.,Biochem Biophys Res Commun 2010; 398:713-718). Many inflammatorycytokines are upregulated in Klf4CN corneas lacking Slurp1 (Table 1;FIG. 2). Slurp1 was down-regulated in HSV-1 infected and LPS-injectedcorneas in the presence of normal levels of Klf4 (FIG. 5). Thus,inflammatory cytokines could selectively inhibit Slurp1 productionwithout affecting Klf4. Consistent with this, treatment of HCLE cellswith IL-4, IL-13 and TNF-αsuppressed SLURP1 production, while IFN-γ didnot affect SLURP1 levels significantly (FIG. 7A). It was determined ifelevated levels of IFN-γ play a role in downregulation of Slurp1 inHSV-1 infected corneas by measuring the Klf4 and Slurp1 levels inabraded or HSV-1 infected WT and IFN-γ knockout (GKO) mice. At 2 DPI,both the extent of Slurp1 downregulation and the nature of leukocyticinfiltrate was comparable between the WT and GKO corneas (FIGS. 7 B, Cand D; Table 2B), suggesting that IFN-γ is not involved indown-regulation of Slurp1 in HSV-1-infected corneas. Thus, inpro-inflammatory conditions, SLURP1 expression is inhibited by cytokinesIL-4, IL-13 and TNF-α, but not IFN-γ.

Example 9 Slurp1 Restricts Neutrophilic Infiltrate inAdenovirus-Infected Corneas

In order to directly test the immunomodulatory role of Slurp1, anadenoviral vector (serotype-5) expressing Slurp1 under the control ofCMV promoter (Adv5-Slurp1) was generated. Wild-type (WT) BALB/c mousecorneas were abraded and infected with control Adv5-Tet-Off vector alone(2×10⁶ plaque forming units (PFU)/cornea) or Adv5-Slurp1 andAdv5-Tet-Off vectors (10⁶ PFU each/cornea). Examination of the infectedeyes at 4 and 10 DPI through a slit-lamp biomicroscope revealed signs ofmild inflammation in corneas infected with Adv5-Tet-Off vector alone,while those infected with Adv5-Slurp1 and Adv5-Tet-Off vectors remainednormal (FIG. 8A). The corneas were harvested from these mice at 4 DPI,and Slurp1 expression in epithelial cells was estimated by QPCR. Inparallel, the nature of leukocytic infiltrate in corresponding cornealstromas was assessed by flow cytometry as above. Slurp1 expression inAdv5-Tet-Off virus infected corneas was reduced to 17% of that in theabraded corneas, and was partially restored in Adv5-Tet-Off andAdv5-Slurp1 co-infected corneas, to 55% of that in the abraded corneas(FIG. 8B). The reduced expression of Slurp1 in Adv5-Tet-Off virusinfected corneas was accompanied by significantly elevated neutrophilicinfiltrate, compared with the small number of neutrophils identified incontrol abraded corneas expressing normal levels of Slurp1 (FIGS. 8C andD). Partial restoration of Slurp1 expression in Adv5-Slurp1 andAdv5-Tet-Off co-infected corneas significantly restricted theneutrophilic infiltrate (FIG. 8B). These results, taken together withthose described above with HSV-1 infected, LPS-injected and Klf4CNcorneas, provide additional evidence of the immunomodulatory role forSlurp1.

It is demonstrated herein that Slurp1 expression is (i) increased uponmouse eyelid opening when the cornea is first exposed to theenvironment, (ii) decreased in inflamed Klf4CN corneas, (iii) criticallydependent on the transcription factor Klf4, (iv) abrogated uponbacterial LPS injection, HSV-1, or adenoviral infection, (v) suppressedby pro-inflammatory cytokines IL-4, IL-13 and TNF-α, and (vi) capable ofrestricting neutrophilic infiltrate in adenovirus infected corneas.Taken together, the results provide the necessary evidence that Slurp1is a key immunomodulatory molecule that contributes to corneal immuneprivilege by suppressing leukocyte infiltration in healthy corneas, andthat is rapidly down-regulated in acute inflammatory conditions to allowprotective inflammation to develop.

The findings demonstrate the relationship between reduced Slurp1 andincreased inflammation. Without being bound by theory, it is possiblethat increased inflammation in the Klf4CN corneas is directly related toreduced Slurp1 expression, as Klf4 modulates the expression of manygenes including Slurp1 (Swamynathan et al., Invest Ophthalmol Vis Sci2011; 52:1762-1769, Swamynathan et al., Invest Ophthalmol Vis Sci 2008;49:3360-3370, Swamynathan et al., Mol Cell Biol 2007; 27:182-194).Moreover, the absence of conjunctival goblet cells (Swamynathan et al.,Mol Cell Biol 2007; 27:182-194) and the loss of corneal epithelialbarrier function (Swamynathan et al., Invest Ophthalmol Vis Sci 2011;52:1762-1769) can also generate pro-inflammatory signals in the Klf4CNocular surface.

The concept of corneal immune privilege arose from the antitheticalnature of inflammation and essential corneal transparence (Niederkorn etal., Ocul Immunol Inflamm 2010; 18:19-23, Azar et al., Trans AmOphthalmol Soc 2006; 104:264-302, Hazlett et al., Ocul Immunol Inflamm2010; 18:237-243, Barabino et al., Prog Retin Eye Res 2012; 31:271-285,Clements et al., Semin Ophthalmol 2011; 26:235-245, Yamanaka et al.,Endocr Metab Immune Disord Drug Targets 2010; 10:331-335, Gronert etal., Exp Eye Res 2010; 91:478-485, Ambati et al., Nature 2006;443:993-997, Cursiefen et al., Proc Natl Acad Sci USA 2006;103:11405-11410, Stuart et al., Invest Ophthalmol Vis Sci 2003;44:93-98, Morris et al., J Immunol 2012; 188:793-799, Jin et al., Am JPathol 2011; 178:1922-1929, El Annan et al., Invest Ophthalmol Vis Sci2010; 51:3418-3423, Tandon et al., Curr Mol Med 2010; 10:565-578). As itis the most anterior part of the eye, the cornea is constantly exposedto various biological, chemical and physical insults. The requirementfor its transparence to ensure proper vision mandates that the cornea bekept free of chronic inflammation in the presence of mild but constantinsults. Corneal cells constitutively express a variety of moleculesthat function to inhibit important components of the inflammatoryresponse. For instance, the avascular nature of the cornea is maintainedin part by the constitutive production of soluble vascular endothelialgrowth factor (VEGF) receptor-1 (sVEGF-R1) and -3 (VEGF-R3) that inhibitthe angiogenic activity of VEGF (Azar et al., Trans Am Ophthalmol Soc2006; 104:264-302, Ambati et al., Nature 2006; 443:993-997, Cursiefen etal., Proc Natl Acad Sci USA 2006; 103:11405-11410). The corneaconstitutively expresses surface molecules such as FAS ligand andProgrammed Death Ligand 1 (PD-L1) that can inhibit or kill infiltratingleukocytes (Stuart et al., Invest Ophthalmol Vis Sci 2003; 44:93-98,Morris et al., J Immunol 2012; 188:793-799, Jin et al., Am J Pathol2011; 178:1922-1929, El Annan et al., Invest Ophthalmol Vis Sci 2010;51:3418-3423, Keir et al., Annu Rev Immunol 2008; 26:677-704), andsecreted molecules such as transforming growth factor beta that potentlyinhibits the function of a variety of inflammatory cells (Tandon et al.,Curr Mol Med 2010; 10:565-578). It is documented herein that Slurp1 is aconstitutively expressed molecule that inhibits inflammatory response inthe cornea.

Without being bound by theory, Slurp1 is a part of the mechanism thatprevents neutrophil infiltration into the normal cornea by one or bothof the pathways depicted in FIG. 9. The first scenario predicts thatSlurp1, which is structurally similar to membrane-tethered uPAR requiredfor neutrophil recruitment in response to bacterial infections (Gyetkoet al., J Immunol 2000; 165:1513-1519, Rijneveld et al., J Immunol 2002;168:3507-3511, Smith et al., Nat Rev Mol Cell Biol 2010; 11:23-36),functions as a soluble scavenger of uPAR ligands and blocks itsfunctions, analogous to the role of soluble VEGFR in blocking cornealangiogenesis (Ambati et al., Nature 2006; 443:993-997) (FIG. 9A, i).Although uPAR-mediated neutrophil recruitment is independent of uPA,(Gueler et al., J Immunol 2008; 181:1179-1189, Connolly et al., Blood2010; 116:1593-1603), other activities of uPAR such as its interactionwith vitronectin and b1-integrin, and bacterial clearance are dependenton uPA (Connolly et al., Blood 2010; 116:1593-1603, Nguyen et al., JCell Biol 1999; 146:149-164, Sidenius et al., J Biol Chem 2002;277:27982-27990, Caiolfa et al., J Cell Biol 2007; 179:1067-1082). Thesecond scenario predicts that Slurp1, which shares the structuralfeatures of a-bungarotoxin and serves as a ligand for the α7nAchRs(Arredondo et al., J Invest Dermatol 2005; 125:1236-1241), suppressesthe release of inflammatory mediators such as TNF-α from macrophages byenhancing α7nAChR mediated responses (Moriwaki et al., Neurosci Res2009; 64:403-412) (FIG. 9A, ii). Normal mouse corneas possess a networkof stromal macrophages and a sparse population of dendritic cells(Brissette-Storkus et al., Invest Ophthalmol Vis Sci 2002; 43:2264-2271,Knickelbein et al., Ophthalmol Eye Dis 2009; 1:45-54, Hamrah et al., IntOphthalmol Clin 2009; 49:53-62), which are likely to express α7nAchR(Kawashima et al., Life Sci 2007; 80:2314-2319), ligation of whichresults in inhibition of cytokine and chemokine release (Grando et al.,J Pharmacol Sci 2008; 106:174-179). In either scenario, when the corneaneeds to mount a rapid immune response to deal with acute infections orsevere chemical or physical insults, downregulation of Slurp1 serves asa molecular switch facilitating further progression of inflammation(FIG. 9B). This role of Slurp1 as a molecular switch regulatinginflammation may not be limited to the cornea; it may serve a similarfunction in the other epithelia where it is abundantly expressed.

An important observation is that the resident population of bonemarrow-derived CD45⁺ cells is altered in Klf4CN corneas that lackSlurp1. The resident leukocyte population in these corneas is not onlylarger, but is also phenotypically distinct from that found in normalcorneas of WT mice. While WT corneal stromas contain mainly CD11b⁺ Gr-1⁻macrophages, those of Klf4CN mice lacking Slurp1 expression contain apopulation of CD11b⁺ Gr-1− cells, a phenotype characteristic ofneutrophils. As eosinophils express CD11b and Gr-1 (Rothenberg et al.,Annu Rev Immunol 2006; 24:147-174), a contribution of eosinophils to theleukocytic population in corneas of Klf4CN mice cannot be ruled out. Thechemokine showing the greatest up-regulation in non-infected Klf4CNcorneas is CXCL5, a chemokine that is induced by IL-1 and TNF-α and is apotent chemoattractant for neutrophils. In addition, two of the threemost up-regulated chemokines in non-infected Klf4CN corneas relative toWT corneas are chemotactic for neutrophils (Table 1). The Klf4CN corneasalso exhibited increased expression of the type 2 cytokines IL-4 andIL-13, and the chemokine CCL11, all known to attract and activateeosinophils. As IL-4 and IL-13 inhibit Slurp 1 expression, the reducedexpression of Slurp1 1 in the Klf4CN mouse corneas could reflect thecumulative effect of the absence of Klf4 and elevated levels of IL-4 andIL-13.

Mock infection of the WT corneas involving abrasion of the cornealepithelium did not reduce Slurp1 expression. These corneas showed few ifany cells expressing a neutrophil phenotype. HSV-1 infection of themouse corneas resulted in a rapid infiltration of leukocytes consistingprimarily of neutrophils, consistent with the previous reports(Hendricks et al., Invest Ophthalmol Vis Sci 1990; 31:1929-1939). It washypothesized that Slurp1 expression would have to be down-regulated topermit neutrophil infiltration into HSV-1 infected corneas. It was foundthat Slurp1 expression is abrogated in the cornea by 24 h after HSV-1corneal infection, and Slurp1 down-regulation was associated with aleukocytic infiltrate predominated by neutrophils.

Interestingly, Slurp1 down-regulation in corneas 24 h after infectionoccurred in the presence of relatively unchanged levels of Klf4,indicating that the early reduction in Slurp1 was not due to adeficiency in Klf4. Slurp1 was virtually abrogated, even though only aportion of corneal epithelial cells were infected by the virus,suggesting that down-regulation of Slurp1 does not require directinfection of corneal epithelial cells. Without being bound by theory, itseems likely that suppression of Slurp1 expression is mediated by asoluble mediator that is induced by HSV-1 infection. IL-4, IL-13 andTNFα-mediated suppression of Slurp1 expression without perturbing Klf4levels is consistent with this likelihood. Even though IFN-γ is rapidlyproduced in the cornea following HSV-1 infection (Hendricks et al., JImmunol 1992; 149:3023-3028), and inhibits Slurp1 expression in vitro(Mastrangeli et al., Eur J Dermatol 2003; 13:560-570), the resultspresented herein demonstrate that IFN-γ is not involved indown-regulation of Slurp1 following corneal infection with HSV-1. Thisis most likely due to overlapping effects of other inflammatorycytokines.

Mutations or deletions in SLURP1 gene are associated with Mal de Meleda,a rare autosomal recessive palmoplantar hyperkeratotic disorder inhumans (Mastrangeli et al., Eur J Dermatol 2003; 13:560-570, Favre etal., J Invest Dermatol 2007; 127:301-308, Chimienti et al., Hum MolGenet 2003; 12:3017-3024, Fischer et al., Hum Mol Genet 2001;10:875-880, Eckl et al., Hum Genet 2003; 112:50-56, Hu et al., J InvestDermatol 2003; 120:967-969, Marrakchi et al., J Invest Dermatol 2003;120:351-355, Ward et al., J Invest Dermatol 2003; 120:96-98). Althoughdiverse inflammatory keratodermas are often associated with ocularsurface defects (Messmer et al., Ophthalmology 2005; 112:e1-6, Mohammadet al., Pediatr Dermatol 2009; 26:113-115, Sonoda et al., Am JOphthalmol 2004; 137:181-183), no such defects have been described sofar in Mal de Meleda patients.

It is demonstrated herein that Klf4 regulates the expression of Slurp1,a key immunomodulatory molecule that is abundantly expressed in thehealthy cornea, and that is rapidly downregulated in pro-inflammatoryconditions. Regulation of expression of Slurp1 is a mechanism in whichKlf4 contributes to maintenance of the corneal homeostasis. SLURP1 isidentified herein as a novel target for therapeutic intervention inmanaging corneal inflammatory disorders of diverse etiologies. SLURP1 isexpressed in other tissues such as skin, oral mucosa, intestine, lung,and cervical epithelium. Thus, SLURP1 can be used in findings andconclusions in other epithelia frequently exposed to similar insults asthe ocular surface.

It is demonstrated herein that Slurp1 modulates corneal fibroblastM/KT-1 cell (i) proliferation, (ii) adhesion to different components ofthe extracellular matrix, and (iii) migration in in vitro gap-fillingassays. It is also demonstrated herein that Slurp1 interacts with uPA,and reduces the amount of cell surface-bound uPA, providing evidencethat Slurp1 serves as a soluble scavenger of uPAR ligands. Finally, itis demonstrated that the expression of SLURP1 in human tears isdependent on the ocular surface health, indicating that our findingswith the mouse model system are generally applicable in humans (seeFIGS. 10-18).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for treating ocular inflammation in a subject,comprising selecting a subject with ocular inflammation, andadministering to the subject a therapeutically effective amount of aSLURP1 polypeptide, or a nucleic acid encoding the SLURP1 polypeptide,wherein the SLURP1 polypeptide comprises an amino acid sequence at least95% identical to the amino acid sequence set forth as amino acids 23-103of SEQ ID NO: 1, thereby treating the ocular inflammation subject. 2.The method of claim 1, wherein the SLURP1 polypeptide comprises an aminoacid sequence at least 95% identical to the amino acid sequence setforth as SEQ ID NO:
 1. 3. The method of claim 1, wherein the SLURP1polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1.4. The method of claim 1, wherein the subject is a human.
 5. The methodof claim 1, wherein the ocular inflammation is keratitis.
 6. The methodof claim 5, wherein the keratitis is bacterial keratitis.
 7. The methodof claim 5, wherein the keratitis is viral keratitis.
 8. The method ofclaim 5, wherein the keratitis results from laser eye therapy, trauma,exposure to ultraviolet light, exposure to chemical stimuli, contactlens wear, corneal transplant, or exposure to a toxin.
 9. The method ofclaim 5, wherein the keratitis is ulcerative.
 10. The method of claim 1,wherein the SLURP1 polypeptide or the nucleic acid encoding the SLURP1polypeptide is administered topically to the eye.
 11. The method ofclaim 1, wherein the SLURP1 polypeptide, or the nucleic acid encodingthe SLURP1 polypeptide, is administered in an ophthalmic solution orointment.
 12. The method of claim 1, further comprising administering anadditional anti-inflammatory agent to the subject.
 13. The method ofclaim 1, comprising administering to the subject a therapeuticallyeffective amount of the polynucleotide encoding SLURP1.
 14. The methodof claim 1, wherein the polynucleotide encoding SLURP1 is operablylinked to a heterologous constitutive promoter.
 15. The method of claim1, comprising administering to the subject a therapeutically effectiveamount of a replication defective adenoviral vector comprising thepolynucleotide encoding SLURP1.
 16. The method of claim 15, wherein theadenoviral vector is administered topically to the eye.
 17. The methodof claim 1, wherein the SLURP1 polypeptide or polynucleotide encodingthe polypeptide is administered in an ophthalmic solution or ointment.18. The method of claim 1, wherein administration of the therapeuticallyeffective amount of the SLURP1 polypeptide, or the nucleic acid encodingthe SLURP1 polypeptide, reduces neutrophil infiltration in the eye. 19.The method of claim 1, wherein the ocular inflammation is caused by abacteria, and wherein the method further comprises administering to thesubject a therapeutically effective amount of an antibacterial agent.